Vol. 124, No. 3 Printed in U.S.A.

JOURNAL OF BACTROLOGY, Dec. 1975, p. 1429-1438

Copyright 0 1975 American Society for Microbiology

Heterologous Deoxyribonucleic Acid Uptake and Complexing with Cellular Constituents in Competent Bacillus subtilis ANNA SOLTYK, D. SHUGAR, AND MIROSLAWA PIECHOWSKA* Institute of Biochemistry and Biophysics, Academy of Sciences, 02-532 Warsaw, Poland

Received for publication 30 June 1975

With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA. Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T*6) DNA is taken up almost as effectively as homologous DNA. Both T6 and T4 DNA interfere only minimally with uptake of homologous DNA; by contrast, T*6 DNA competes with homologous DNA as effectively as the latter itself. These results indicate that the glucose residues in the T-even phage DNA, located in the large groove of the DNA helix, reduce affinity for cellular receptors, leading to low binding of T6 DNA. The latter DNA is considerably less degraded by extracellular nucleases than homologous DNA, thus excluding enzymatic hydrolysis as the source of poor uptake. Affinity of DNA for competent cells was also evaluated by the formation, and detection in a CsCl density gradient, of complexes of DNA with cellular constituent(s). Such complexes, similar to those previously observed with transforming DNA, are formed by E. coli DNA and T*6 DNA; in reconstruction experiments the denatured forms of these same DNA samples form complexes when added to the cells before lysis. T6 DNA, on the other hand, does not form such a complex. The possible role of such complexes in transport of DNA to the cell interior is discussed. The uptake of deoxyribonucleic acid (DNA) by competent bacterial cells has frequently been considered a rather nonspecific process (7, 20, 24, 28) since competent cultures of Diplococcus pneumoniae, Bacillus subtilis, and streptococci bind to a similar extent both homologous and heterologous DNA, whether the latter is of bacterial, phage, or mammalian origin (2, 12, 15). Some reservations have, however, been expressed with regard to this view, and there are reported instances of discrimination against heterologous DNA by competent cultures of Haemophilus influenzae (7, 20, 21). Notwithstanding a recent detailed investigation by Scocca, Poland, and Zoon (22), the mechanism by means of which H. influenzae discriminates between DNAs of different origins remains to be clarified. In a previous study on the fate of heterologous DNA in competent B. subtilis (17), the uptake of phage T6 DNA was observed to be surprisingly low. This prompted us to examine in more detail the behavior in this system of several heterologous DNAs, relative to that of a homologous preparation, with respect to uptake, affinity for presumed cell receptors for transforming DNA (usually based on measurements of competition for uptake between

homologous and heterologous DNA), and possible binding of denatured forms with cellular constituents exhibiting specific affinity for denatured DNA. Complexing of denatured DNA with some cellular constituent(s) of B. subtilis was previously noted with homologous transforming DNA (16). MATERIALS AND METHODS Bacterial strains. The B. subtilis strains employed, all from the laboratory of M. S. Fox of the Massachusetts Institute of Technology, included B. subtilis 168 thy- trp- as the recipient, and B. subtilis 168+ as the donor, of transforming DNA, and B. subtilis 168 thy- trp+ as the donor of radioactively labeled transforming DNA. Sources of heterologous DNA included the thymine-dependent strain Escherichia coli 15T- and phages T4 and T6, provided by Irena Pietrzykowska of this Institute. The phages were cultivated on E. coli BB, obtained from W. Szybalski of the University of Wisconsin. Nonglucosylated phage T6 DNA (T*6 DNA) was prepared from phage T6 cultivated on the uridine diphosphoglucose phosphorylase-defective mutant of E. coli B-4° Luria no. 56 (6), provided by S. E. Luria of the Massachusetts Institute of Technology. The nonglucosylated phage T6 (T*6) titer was determined with the aid of the strain Shigella dysenteriae, supplied by N. Symonds of the University of Sussex, England. Absence of glucosylation was confirmed by

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SOLTYK, SHUGAR, AND PIECHOWSKA

J. BACTERIOL.

the 300-fold lower phage titer when cultivated on E. coli BB. Micrococcus luteus was a strain originally obtained from the ATCC collection. Competent cultures. Competent cultures of' the recipient strain were prepared as elsewhere described by Piechowska and Fox (16) in media containing thymine (20 ug/ml). For competition experiments, the recipient was cultivated on a medium containing 0.16 mCi of ["4C]thymine (from UVVVR, Czechoslovakia) per mmol of thymine. In experiments designed to test the interaction of heterologous DNA with cellular constituents, the recipient was labeled with ["4C Ithymine by use of a medium containing 0.32 mCi per mmol of thymine. The density of the recipient culture was 2 x 108 cells/ml. Competence, measured by the number of transformants for the tryptophane synthesis marker, varied from 0.5 to 1%. Preparation of DNA. (i) Transforming DNA. Transforming DNA was isolated from B. subtilis 168+, cultivated in Pennassay broth (Difco antibiotic medium 3), by the procedure of Saito and Miura (19), modified as follows. After treatment of the cells with lysozyme, freezing and thawing were omitted; lysis was terminated with Sarcosyl NL (Geigy Industrial Chemicals, Ardsley, N.Y.) at a final concentration of 0.25% and heating of the sample for 15 min at 62 C. Ribonucleic acid (RNA) was removed by treatment of the mixture with ribonuclease (RNase) I (Worthington Biochemical Corp., Freehold, N.J.) and RNase T, (Boehringer GmbH, Mannheim, G.F.R.). (ii) T4, T6, and T*6 DNA. T4, T6, and T*6 DNA preparations were obtained by phenol deproteinization of phages purified by differential centrifugation, and centrifugation in preformed CsCl density gradients as described by Thomas and Abelson (27). (iii) Calf thymus DNA. Calf thymus DNA was prepared from deoxyribonucleoprotein isolated by the method of Zubay and Doty (cited in Sonnenberg and Zubay, reference 23). The nucleoprotein was deproteinized with phenol, and the DNA was precipitated with ethanol and dissolved under conditions similar to those for the bacterial DNA. Labeled DNAs. 9H-labeled DNA from B. subtilis, phage T6 and phage T*6, B. subtilis "4C-labeled DNA, E. coli 2H, 3H-labeled DNA, E. coli 2H, '4C labeled DNA, and M. luteus 92P-labeled DNA were all prepared as previously described (17). E. coli "4C-labeled DNA was isolated from E. coli 15T- which was cultivated in a medium containing (in 1 liter): K2HPO4, 11.2 g; KH2PO4, 4.8 g; (NH4)2SO,4, 2.0 g; supplemented with MgSO4 to a concentration of 10-3 M, FeSO4 to a concentration of 3.3 x 10-" M, glucose to a concentration of 0.5%, vitamin-free Casamino Acids (Difco) to 1%, thymine to 3 14g/ml, and [4C ]thymine (UVVVR, Czechoslovakia) to 80 mCi per mmol of thymine in the medium. The cells were lysed and the DNA was purified as described for B. subtilis labeled DNA, but with the use of sodium lauryl sulfate (British Drug Houses, Poole, England) in place of Sarcosyl. All the radioactive DNA preparations were purified by fractionation of cell lysates on CsCl density gradients. The fractions containing the DNA were pooled and dialyzed against a solution of SSC (0.15 M NaCl

plus 0.015 M sodium citrate) at pH 7. DNA concentrations were determined by their optical densities at 260 nm. The specific activities of the various preparations are given in the experiments described. Measurement of DNA uptake. To 0.8 or 1.9 ml of' a competent culture was added 0.2 or 0.1 ml of' 3H-labeled DNA at the desired concentration. The culture was incubated with shaking at 30 C for 15 min. DNA uptake was terminated by addition of DNase I (Worthington Biochemicals Corp., Freehold, N.J.) to a concentration of 25 Ag/ml and incubation f'or 0.5 min at 37 C. This was followed by addition of' an equal volume of cold 0.15 M NaCl plus 0.1 M EDTA (ethylenediaminetetraacetate), pH 8. The cells were collected by centrifugation at 4 C, washed twice with 0.15 M NaCl plus 0.1 M EDTA, pH 8, and suspended in 0.5 ml of the same solution. To this suspension was added 0.1 ml of albumin (bovine albumin f'raction V powder, Pentex) at a concentration of 1 mg/ml and 1 volume (i.e., 0.6 ml) of 10% trichloroacetic acid. The sample was kept in an ice bath for 20 min and then filtered under reduced pressure through a glass filter (glass-fiber paper GF/C, Whatman). The precipitate was rinsed with 5% trichloroacetic acid, alcohol, and ether, dried and counted. Uptake values were corrected for radioactivity bound by a control culture incubated with the same DNA sample previously degraded with DNase I. Cell lysates for density gradient centrifugation. After DNA uptake by a recipient cell suspension (see above), lysozyme (Sigma Chemical Co., St. Louis, Mo.) was added to a concentration of' 7 mg/ml. Incubation was then conducted for 30 to 40 min at 2 C, interrupted four times to warm the samples to 37 C for 2-min intervals. Sarcosyl NL was added to a concentration of 1% and the sample was warmed to 10 C for 1 min. The lysate was then treated overnight with nuclease-free Pronase (Calbiochem) added to a concentration of 1 mg or 0.1 mg/ml. Fractionation on CsCl gradients was always preceded by two dialysis steps, the first against 0.15 M NaCl plus 0.01 M EDTA, pH 8 and the second against 0.15 M NaCl plus 0.001 M EDTA, pH 8. CsCl gradient fractionation. Gradients were prepared as described by Piechowska and Fox (16). After centrifugation, the bottom of the tube was pierced and 3-drop fractions were collected on 21-mm-diameter Whatman 3MM paper disks and, in this form, precipitated with trichloroacetic acid before counting. Radioactivity measurements. Counting was done with a Packard Tri-Carb scintillation counter. The scintillation fluid consisted of 3 g of 2,5diphenyloxazole and 0.1 g of 1,4-bis-(5-phenyloxazolyl)benzene (A.G. Fluka, Zurich) in 1 liter of toluene. Specific activities of DNA samples were determined on GF/C filters under optimal conditions for a given label. Sedimentation coefficients of DNA preparations. Sedimentation coefficients of DNA preparations were determined in a Spinco model E instrument fitted with ultraviolet optics using a 30-mm cuvette with a 40 Kel-F centerpiece, at 20 C, at 16,000 rpm for the high-molecular-weight samples and at 44,000 rpm for calf thymus DNA. The mean values of the sedimenta-

1430

VOL. 124, 1975

HETEROLOGOUS DNA UPTAKE

1431

tion constants were corrected to give the s2,.w values DNA, and good uptake of T*6 DNA. The mean described by Studier (25). These were not extrapo- values for all experiments, with the use of lated to zero DNA concentrations; all preparations different recipient cultures and different DNA were centrifuged at a low concentration of 17 Ag of DNA/ml. Molecular weights were calculated with the preparations, although exhibiting rather wide variations, were in accord with the foregoing. aid of the equations of Studier (25). Denatured DNA. Denatured DNA for use in The variations of individual measurements reconstruction experiments or as a marker in CsCl from the mean (Table 1) are of particular gradients was obtained by thermal denaturation: significance in relation to the DNA of phage T6 0.5-ml samples containing 10 Mg of DNA per ml in and suggest it is perhaps not taken up at all. In 0.1x SSC were heated at 100 C for 6 min and then view of the known dependence of uptake on cooled rapidly in an ice bath. molecular weight when the latter is of the order as

RESULTS

Uptake of labeled DNA. Uptake of heterolo3H-labeled DNA from phage T6, phage T*6, and E. coli was compared with that for tritiated B. subtilis transforming DNA, with results illustrated in Table 1. Typical experiments, in which samples of the same recipient culture were used to compare uptake of two different DNAs, showed that E. coli DNA was taken up about one-half as effectively as homologous DNA, very low (if any) uptake of T6 gous

of 16 x 106 or lower (1), it should be emphasized that the molecular weight of the T6 DNA referred to in experiment 6 of Table 1 was 23 x 106, hence sufficiently high to exclude this as a factor in the very low observed binding. In those experiments where binding of the T6 DNA label by cells was detected, prolongation of treatment with DNase I (from the usual 0.5 min to 40 min) removed 60% of the radioactivity from the cells; under the same conditions with B. subtilis DNA, only 20% of the activity was removed. In view of the marked difference in behavior

TABLE 1. Uptake of 8H-labeled DNA by B. subtilisa Sp act

Expt no.

Source of 'H-labeled DNA

No. of trials

(counts/min

DNA concn

per Ag

(jsg/ml)

x

Bacillus subtilis (2) Escherichia coli B. subtilis (2) E. coli B. subtilis (2) E.coli B. subtilis (1) T6 (1) B. subtilis (1) T6 (1) T6 (2) T*6 (1)

1 2

3 4

5 6

9

B. subtilis

5

E. coli

10

T6

3

T*6

10-')

8.5 7.9 8.5 7.9 8.5 7.9 3.2 2.9 3.2 2.9 1.6 1.6

3.2 8.5 4.2 7.9 2.9 1.6 4.3 9.0

1.6 0.8

Normalized DNA uptake/ 4 x 10' cells

uptake

(Mg x 102)

°

1.23 0.58 1.17 0.44 0.94 0.60 0.74 0.10 1.04 0.18 0 0.71

100 48 100

1.4 1.4 1.4 1.4 1.4 1.4 1.8 1.8 1.8 1.8 2.8 1.4

DNA utk

37 100 64 100 14 100 17

1-2

1.38

1.4

0.62 ± 0.15

45

1-3.3

0.095 ± 0.095

7

1-1.4

1.25 ± 0.48

90

0.51b

100

a Uptake of various 'H-labeled DNAs by 1.9-ml suspensions of competent cells, measured as described in the text. Different recipient cultures were employed in individual experiments; hence uptake of each heterologous DNA preparation was measured relative to that of homologous DNA. Different DNA preparations (indicated by number) from the same source were also used, with specific activities as indicated in column 4. Uptake values were normalized to a DNA specific activity of 4 x 10' counts/min per ug. For the various DNA preparations the OD,2/OD2.. ratios varied from 1.6 to 2.3. b Mean values.

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SOLTYK, SHUGAR, AND PIECHOWSKA

J. BACTERIOL.

of phage T6 DNA relative to the other DNA preparations, uptake of this DNA was examined at different incubation temperatures. At 20, 30, and 37 C, uptake of T6 DNA corresponded to 484, 835, and 1,120 counts/min, respectively, per 3.8 x 108 cells. The validity of the findings for the DNA preparations more effectively taken up was c,E 101 further examined with the aid of calibration curves for the DNA of T*6 and B. subtilis, shown in Fig. 1. These curves show the approxi6mately 30% lower uptake of T*6 DNA at con4centrations above 3 gg/ml, corresponding to the plateau of the calibration curve. It should be 2emphasized that both DNA samples were of high molecular weight, 10.3 x 106 for B. subtilis 10 20 30 40 50 60 0 80 90 DNA and 38.8 x 106 for T*6 DNA. Effect of incubation of DNA with competent cells on its sedimentation properties. B Conceivably the poor uptake of T6 DNA could 1015 min be due to a reduction in molecular weight resulting from hydrolysis by a nuclease(s) at the cell surface. This was tested by comparing the sedimentation properties of B. subtilis 14Clabeled DNA and phage T6 3H-labeled DNA 4before, and after, incubation with competent cells under the same conditions as employed for 2 measurement of uptake. From Fig. 2 it will be seen that sedimentation in a sucrose gradient 10 20 30 40 50 60 70 80 90 shows no marked change in rate of sedimentafraction number tion of T6 DNA; there is, in fact, less damage FIG. 2. Sedimentation of T6 3H-labeled DNA (A) than in the case of homologous DNA. and B. subtilis 'IC-labeled DNA (A) at time 0 (A) and after 15-min (B) exposure to competent recipients. To 0.9 ml of recipient cells was added 0.55 Mg of 2 T6 'H-labeled DNA (1.6 x 10' counts/min per /g) 0 0 and 0.5 jug of B. subtilis 168 thy- "C-labeled DNA ° 6(3.2 x 10' counts /min per Mg) in 0.1 ml of SSC. The suspension was divided into two 0.5-ml samples, which were cooled, and to each was added 50 ul of 0.25 4M EDTA, pH 8, followed by removal of the cells by centrifugation. A 0.2-ml amount of supernatant was deposited on 4.9 ml of 5 to 20% (wt/vol) linear sucrose gradients in 1 M NaCI plus 0.01 M tris(hydroxymethyl)aminomethane plus 0.005 M EDTA, pH 7.5, in AA

7

1

2

3

4

5

6

7

8

DNA concentration ( g/mI)

FIG. 1. Uptake of B. subtilis 168 thy- 3H-labeled DNA (O) and phage T*6 3H-labeled DNA (0) by B. subtilis competent culture as a function of DNA concentration. Samples were prepared as described for 0.8 ml of recipient cells. Specific activities of DNA preparations were 2.55 x 105 counts/min per Ag of B. subtilis DNA, and 2.3 x 105 counts/min per gg of Tr6 DNA. The figures for uptake were standardized with respect to the specific activity of T*6 DNA, so that 1,000 counts/min = 0.62 x 10-2 ug/1.6 x 10' cells. Molecular weights were 10.3 x 10 and 38.8 x 106 for B. subtilis and T*6DNAs, respectively.

nitrocellulose tubes, and centrifuged in a Spinco SW 50.1 rotor for 110 min at 39,000 rpm at 20 C. Fractions were collected on VWhatman 3MM paper and precipitated with trichloroacetic acid, and radioactivity was counted as described in the text.

Competition experiments. To better characterize the specificity of interaction of DNA with cellular receptors, the competitive inhibition of transforming 3H-labeled DNA uptake by nonlabeled heterologous DNA from different sources was compared with the competition due to homologous DNA, using saturating concentrations of labeled DNA. To this end, the phage DNA samples employed were further characterized. For all three

VOL. 124, 1975

HETEROLOGOUS DNA UPTAKE

1433

preparations employed as competitive inhibitors of uptake, the value of optical density at 260 nm/optical density at 230 nm (OD2,,/OD2,,0) was close to 2.2 or higher, hence better than values normally reported in the literature for such purified phage DNA (27) and testifying to a low protein content. Molecular weights varied from 8 x 10' to 23 x 10' for T4 DNA, and 40 O x 10to44 x lO'forT6 and T*6 DNA. Inhibition of B. subtilis 3H-labeled DNA uptake by increasing concentrations of heterolo- + 4 gous T4, T6, and T*6 DNA preparations is illustrated by Fig. 3. A sevenfold excess of T4 or T6 DNA reduced transforming DNA uptake by only 10 or 30%, respectively. The control curve for competition by homologous DNA exhibited the decrease in uptake expected for dilution of 7 5 9 11 13 label by cold DNA. competing DNA (1g/ml ) Influence of T6 DNA on transformation. 4. Effect of T6 DNA (A) and calf thymus DNA At a constant concentration of transforming (0)FIG. on transformation to the tryptophane synthesis DNA, and with increasing concentration of T6 marker (trp+) by B. subtilis 168+ DNA. To a compeDNA, the number of transformants exhibiting tent culture were added, simultaneously, tranforming the tryptophan synthesis marker decreased by DNA to a concentration of 0.25 ,g/ml and competing only 30% with a 40-fold excess of T6 DNA. By DNA to concentrations indicated in the figure. DNA contrast, a control with the use of calf thymus uptake proceeded for 20 min at 37 C and was termiDNA exhibited normal competition propor- nated by a 5-min treatment with DNase, 20 gg/ml. tional to the dilution of transforming DNA by The number of trp+ transformants was determined by plating on the selective medium of Mahler (14). the competing molecules (Fig. 4). are expressed as percentage of the number of Affinity of heterologous DNA for cellular Results transformants obtained in the absence of competing constituents. Deuterium- and tritium-labeled DNA. E. coli DNA was added to a ["C ]thyminelabeled culture of competent cells. After DNA a CsCl density gradient as described above. The uptake, the cells were lysed and fractionated on expected relative locations of native recipient DNA, and native and denatured heavy E. coli DNA, were determined by centrifugation of a control mixture of purified samples with M. luteus DNA, the latter of which served as a marker both in the control and the cell lysate (Fig. 5A). The cell lysate, after uptake of E. coli DNA, exhibited the expected DNA recipient band; but the donor DNA appeared as a broad irregular band with a density lower than that for purified E. coli 2H,3H-labeled DNA (Fig. 5B). In analogous experiments with T*6 3H-labeled DNA, the molecules taken up were also found in an irregular "too light" band (results not shown). These results testify to binding of 7 3 9 17 19 23 5 13 15 21 with a cellular constituent(s), leading to a DNA compet,ng DNA (,O/ml) decrease in density in the CsCl gradient, since FIG. 3. Competition of cold T4 DNA (0), T6 DNA no such complex is observed in the absence of (A), T6 DNA (0) and B. subtilis trasforming DNA the bacterial cells (16). (K) for uptake of B. subtilis 3H-labeled DNA. To 0.8 The specificity of complex formation was also ml of a competent recipient culture was added 0.2 ml examined in reconstruction experiments with E. of a solution containing 2 ,ug of 3H-labeled DNA and coli and T6 DNA in comparison to homologous different quantities of cold competing DNA in SSC. Incubation and further treatment was as described in B. subtilis DNA. Equal quantities of denatured the text for measurements of DNA uptake. Specific E. coli "4C-labeled DNA and denatured B. activity of B. subtilis 3H-labeled DNA was 7 x 10' subtilis 3H-labeled DNA were added to a suscounts/min per gg. pension of recipient cells at 2 C. To a similar ,

.n

1

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SOLTYK, SHUGAR, AND PIECHOWSKA

J. BACTERIOL.

suspension

Q.

A

1

20^

c

L)

^

16

co

N-Bs

DH-Ec X1

600 ML

8

400

41

2

30

50

40

60

70

N-Bs i

250-

80

B

200D -Ec N-Ec

*.. 150Z

ML 100100-

t

50-

20

30

~ ~ traction number FIG. 5. Complex formation between E. coli DNA and cellular constituent(s). (A) Relative buoyant densities on a CsCI gradient, pH 8, of DNA samples

were

added

equal quantities of dena-

tured T6 3H-labeled DNA and denatured B. subtilis "4C-labeled DNA. The cell suspensions were lysed and fractionated on CsCl gradients in the same manner as after DNA uptake. In both experiments the B. subtilis DNA used as control was found in a broad band, pointing to its binding by some cell component(s) which leads to an apparent decrease in its density (Fig. 6A and 6C). The heterologous E. coli DNA exhibited analogous behavior (Fig. 6A). By contrast, the T6 DNA banded at the density expected for its denatured form (Fig. 6C). The expected positions for denatured and native DNA in the gradient, utilizing control mixtures of DNAs, are shown in Fig. 6B and 6D. The position of denatured E. coli DNA was established by determining the difference in density between denatured and native DNA in a control mixture centrifuged in a gradient at the pH utilized in the experiment, and adding this difference to the density of E. coli DNA reported in the literature (26). DISCUSSION The results herein reported demonstrate that discrimination against some types of heterologous DNA is exhibited by competent B. subtilis, and that this is particularly pronounced in the case of phage T6 (and probably also T4) DNA. In view of the known dependence of DNA uptake by competent cells on molecular weight (1), it should be pointed out that this factor may be excluded in the present work. This follows from the fact that T6 DNA, with a molecular weight of 23 x 106, was most poorly taken up, and T*6 DNA, with a molecular weight of 39 x 10 , was taken up less effectively than homologous DNA with a molecular weight of 10 x 106. The very poor binding of T6 DNA was not due to any specific degradation by extracellular nucleases (but does not exclude the possibility of some degradation by exonucleases, since the resulting acid-soluble products would not show up in Fig. 2). From Fig. 2 it is clear that T6 DNA was more resistant to such degradation than homologous DNA, in agreement with the known poorer susceptibility of T-even phage DNA to some nuceasebenzy (3 1,3)In fAct, some nuclease enzymes (13, 18, 31) In fact poorer hydrolysis of T6 DNA by the DNase I employed to terminate uptake might possibly

employed: (ML) native M. luteus 32P-labeled DNA; (NH-Ec) native E. coli 2H, 4C labeled DNA; (DH-Ec) denatured E. coli 2H,3H-labeled DNA; (N-Bs) native B. subtilis 3H-labeled DNA. (B) CsCI pH 8 density be considered as a source of error in the quantigradient analysis of a cell lysate prepared 0.5 min after uptake of E. coli 2H,3H-labeled DNA by a 4C-labeled competent culture. To a culture of 3.8 x 108 cells/ml was added E. coli 2H,3H-labeled DNA (2.8 x 105 counts/min per ug) to a concentration of 1 gg/ml. See Materials and Methods for conditions of

DNA uptake, preparation of cell lysate, and fractionation on CsCI gradient. Letters and arrows point to expected positions of various DNA samples. Radioactivities are denoted as: A, 3H; A, 14C; 0, 2p.

VOL. 124, 1975

HETEROLOGOUS DNA UPTAKE

tative determination of such uptake and suggests that adsorption may, instead, be involved. Adsorbed molecules may also conceivably be

1435

degraded by DNase I to a lesser extent because of the more difficult accessibility of bound DNA to the enzyme, a factor of no significance in the

x en,

traction number

FIG. 6. Distribution on a CsCI density gradient of various denatured DNAs after addition to a suspension of recipient cells. To 3.8 x 10' cells, suspended in 0.5 ml of 0.15 M NaCI plus 0.01 M EDTA at 2 C, was added (A) 0.03 Mg of denatured B. subtilis 'H-labeled DNA (1.6 x 10' counts/min per gg) and 0.03 Mg of denatured E. coli "C-labeled DNA (6.2 x 104 counts/min per sg) in 0.1 ml of SSC, and (C) 0.08 jig of denatured B. subtilis "C-labeled DNA (9.3 x 104 counts/min per Mg) and 0.08 Mg of denatured T6 'H-labeled DNA (4.3 x 10' counts/min per Mg) in 0.1 ml of SSC. The cells were lysed as described in the text, and CsCI fractionation was carried out at (A) pH 11.2 and (C) pH 10.8. Frames (B) and (D) exhibit the relative densities of the DNA preparations used in this experiment. Abbreviations: (N-B&) Native B. subtilis "IC-labeled DNA; (D-Bs) denatured B. subtilis "IC-labeled DNA; (N-T6 native T6 'H-labeled DNA; (D-T6) denatured T6 'H-labeled DNA. All of these were added to a CsCl solution and centrifuged. Arrows and abbreviations point to expected locations of individual DNAs. In the gradients (A) and (C), expected positions for each DNA were established by refractometric measurements of the density gradient. Differences in density between native and denatured DNA at a given pH were determined from the control mixture. 7he density of native E. coli DNA was taken as 1.710 g/ml (26). Radioactivities were denoted by: A, 'H; A, 14C.

1436

J. BACTERIOL.

SOLTYK, SHUGAR, AND PIECHOWSKA

case of DNA which undergoes good uptake. The observed temperature dependence of the extent of binding of T6 DNA, which is similar to that for homologous DNA and is a property characteristic of DNA uptake by competent cells (7, 20), is in apparent contradiction with the foregoing argument. Of some interest is the competitive inhibition of homologous DNA uptake by phage DNA, which appears to be related to the extent of glucosylation of 5-hydroxymethylcytosine residues (11). A sevenfold excess of T4 DNA (in which each 5-hydroxymethylcytosine residue is monoglucosylated) decreased homologous DNA uptake by 15%. For T6 DNA (where 25% of the 5-hydroxymethylcytosine residues are linked to two glucose molecules, and 75% are nonglucosylated), uptake was decreased by 30%. By contrast, nonglucosylated T*6 DNA reduced homologous DNA uptake as effectively as the latter, i.e., about 85%. If the foregoing results are interpreted in terms of differences in affinity for some cellular receptor(s), it would follow that the presence of glucose residues, located in the large groove of the DNA helix, as well as the distribution of these residues along the helix, in some way sterically hinders interaction with such receptors. The nature of the base residues appears to be of lesser importance, since competent B. subtilis almost equally as readily takes up T*6 DNA (34% guanine plus 5-hydroxymethylcytosine) as homologous DNA (43% guanine plus cytosine) (26) and also takes up transfecting DNA from a number of phages which contain 5-hydroxymethyluracil in place of thymine (29). The relative abilities of the various DNAs to complex with cellular constituents(s), described above, are of interest in relation to the previously observed formation of such a complex by homologous DNA taken up (16). It is conceivable that such complexes may be involved in the transport of DNA through the cell membrane, as well as in recombination. If this were to be the case, then the analogous complex formation observed in this study with E. coli DNA, which is not integrated in the recipient chromosome (17), argues rather against any role of such a complex in integration. Several observations suggest a possible role for such complexes in DNA uptake. Previous findings (16, 17), and the present results, are consistent in showing that the complexes formed between homologous or heterologous DNA with cellular constituents are sensitive to Pronase and the pH of the CsCl gradient, but without release of free DNA. DNA release does

not occur even in 1% Sarcosyl at 37 C, but does so at60Cin4MNaCl (16, 17), orat70Cin0.15 M NaCl, or by phenol treatment at pH 8 at room temperature (unpublished data), and the liberated DNA is in the denatured form. The properties of these complexes are reminiscent of cell envelope proteins relatively insensitive to proteolytic enzymes and detergents (5, 32). This is in accord with the binding of transforming DNA by B. subtilis cell membranes, revealed by means of fractionation of cell lysates in a Renografin gradient (3) and by autoradiography

(30).

Some light on the possible function of these complexes is provided by the "reconstruction" experiments described above. This procedure gave complexes with E. coli DNA and T*6 DNA, both of which are readily taken up, but not with T6 DNA, which is poorly taken up. It appears reasonable to assume that the cellular constituent(s) involved in such complex formation plays some role in DNA transport, an assumption supported by the observation of Eisenstadt et al. (4) on a binding activity for denatured DNA in competent, but not in noncompetent, B. subtilis cultures. If such is indeed the case, then the use of T6 DNA along with a DNA that undergoes high uptake might serve as an assay system for a transport protein(s) in a procedure designed to isolate this protein, which, according to some views (8-10), should also exhibit nucleolytic activity. If so, then the lower susceptibility of glucosylated DNA to such nuclease(s) could also account for its poor uptake. In view of the interest in DNA-protein complexes (2a, 3, 4, 16), it is worth noting that: (i) no such complex is found in a system containing native or denatured DNA plus all other constituents except cells (16); (ii) the properties of the complex are reminiscent of complexes with membrane proteins (5, 32); (iii) the specificity of complex formation depends on the nature of the DNA, as shown in this study. This points to involvement in the complex of a cellular constituent(s) and not of added proteins such as lysozyme or Pronase (2a). Complex formation most likely occurs during DNA uptake, but, as is usually the case in analyses based on cell fractionation techniques, it is difficult to exclude such complex formation during the lysis process. The low extent of binding of T6 DNA, and the absence of the denatured form of this DNA in cell lysates, contrasts sharply with the absence in cell lysates of the native forms of DNAs which undergo good uptake (16,17). This is

HETEROLOGOUS DNA UPTAKE

VOL. 124, 1975

consistent with the existence of two types of DNA binding by competent B. subtilis, that with high efficiency being accompanied by disruption of the DNA secondary structure, as for uptake by D. pneumoniae (9). Although the results of this investigation have made it possible to formulate an interpretation for the very low binding of T6 DNA in terms of its characteristic structural properties, the source of the smaller differences in uptake between E. coli DNA on the one hand, and T*6 and B. subtilis DNA on the other, remains to be clarified. Furthermore, it should be noted that discrimination against heterologous DNA is not limited to the B. subtilis system. Similar results have since been obtained with competent Streptococcus challis (la). From the observations of Scocca et al. (22) referred to above, and supported by findings in another laboratory (S. Goodgal, personal communication), the H. influenzae system apparently exhibits even stricter discrimination against heterologous DNA, although no interpretation for this is as yet forthcoming. One possibility which has not hitherto been considered is the possible involvement of some specific DNA-linked residual protein as the source of differences in DNA uptake; but this might require more stringent methods of DNA purification. ACKNOWLEDGMENTS We are indebted to all who kindly made available bacterial strains. M.P. would like to thank G. Venema, W. Konings, and H. Joenje for useful discussions. This investigation was carried out as project 09.3.1 of the Polish Academy of Sciences, and profited from the partial support of the Agricultural Research Service, U.S. Department of Agriculture.

LITERATURE CITED 1. Cato, A., Jr., and W. R. Guild. 1968. Transformation and DNA size. I. Activity of fragments of defined size and a fit to a random double cross-over model. J. Mol.

Biol. 37:157-178. la. Ceglowski, P., P. G. Fuchs, and A. Sottyk. 1975. Competitive inhibition of transformation in group H Streptococcus strain Challis by heterologous deoxyribonucleic acid. J. Bacteriol. 124:1621-1623. 2. Chen, K. C., and A. W. Ravin. 1966. Heterospecific transformation of pneumococcus and streptococcus. Relative efficiency and specificity of DNA helping effect. J. Mol. Biol. 22: 123-134. 2a. Davidoff-Abelson, R., and D. Dubnau. 1973. Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded deoxyribonucleic acid. J. Bacteriol. 116:146-153. 3. Dooley, D. C., and E. W. Nester. 1973. Deoxyribonucleic acid-membrane complexes in the Bacillus subtilis transformation system. J. Bacteriol. 114:711-722. 4. Eisenstadt, E., R. Lange, and K. Willecke. 1975. Competent Bacillus subtilis cultures synthesize a denatured DNA binding activity. Proc. Natl. Acad. Sci. U.S.A. 72:323-327.

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5. Ferro-Luzzi Ames, G., E. N. Spudich, and H. Nikaido. 1974. Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406-416. 6. Hattman, S., and 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. 7. Hayes, W. 1969. Genetics of bacteria and their viruses, 2nd ed. John Wiley and Sons Inc., New York. 8. Joenje, H., and G. Venema. 1975. Different nuclease activities in competent and noncompetent Bacillus subtilis. J. Bacteriol. 122:25-58. 9. Lacks, S. 1962. Molecular fate of DNA in genetic transformation of pneumococcus. J. Mol. Biol. 5:119-131. 10. Lacks, S., B. Greenberg, and M. Neuberger. 1974. Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc. Natl. Acad. Sci.

U.S.A. 71:2305-2309. 11. Lehman, I. R., and E. A. Pratt. 1960. On the structure of the glucosylated hydroxymethylcytosine nucleotides of coliphages T2, T4 and T6. J. Biol. Chem. 235:3254-3259. 12. Lerman, L. S., and L. J. Tolmach. 1957. Genetic transformation. I. Cellular incorporation of DNA accompanying transformation in Pneumococcus. Biochim. Biophys. Acta 26:68-82. 13. Lichtenstein, J., and S. S. Cohen. 1960. Nucleotides derived from enzymatic digest of nucleic acids of T2, T4, T6 bacteriophages. J. Biol. Chem. 235:1134-1141. 14. Mahler, I. 1968. Procedures for Bacillus subtilis transformation, p. 846-850. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12B. Academic Press Inc., New York. 15. Pene, J. J., and W. R. Roming. 1964. On the mechanism of genetic recombination in transforming Bacillus subtilis. J. Mol. Biol.. 9:236-245. 16. Piechowska, M., and M. S. Fox. 1971. Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bacteriol. 108:680-689. 17. Piechowska, M., A. Soltyk, and D. Shugar. 1975. Fate of heterologous deoxyribonucleic acid in Bacillus subtilis. J. Bacteriol. 122:610-622. 18. Richardson, C. C. 1966. Influence of glucosylation of deoxyribonucleic acid on hydrolysis by deoxyribonucleases of Escherichia coli. J. Biol. Chem. 241: 2084-2092. 19. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 20. Schaeffer, P. 1964. Transformation, p. 87-144. In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. 5. Academic Press Inc., New York. 21. Schaeffer, P., R. S. Edgar, and R. Rolfe. 1960. Sur l'inhibition de la transformation bacterienne par des desoxyribonucleates de composition variees. C. R. Soc. Biol. 154:1978-1983. 22. Scocca, J. J., R. L. Poland, and K. C. Zoon. 1974. Specificity in deoxyribonucleic acid uptake by transformable Haemophilus influenzae. J. Bacteriol.

118:369-373. 23. Sonnenberg, B. P., and G. Zubay. 1966. Isolation of nucleohistone, p. 600-614. In G. L. Cantoni and D. R. Davis (ed.), Procedures in nucleic acid research. Harper and Row Publishers, New York 24. Spizizen, J., B. E. Reilly, and A. H. Evans. 1966. Microbial transformation and transfection. Annu.Rev. Microbiol. 20: 371-400. 25. Studier, F. W. 1965. Sedimentation studies of size and shape of DNA. J. Mol. Biol. 11:373-390. 26. Szybalski, W. 1968. Use of cesium sulphate for equilib-

1438

SOLTYK, SHUGAR, AND PIECHOWSKA

J. BACTERIOL.

rium density gradient centrifugation, p. 330-360. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12B. Academic Press Inc., New York. 27. Thomas, C. A., and J. Abelson. 1966. The isolation and characterisation of DNA from bacteriophage, p. 553-561. In G. L. Cantoni and D. R. Davies (ed.), Procedures in nucleic acid research. Harper and Row Publishers, New York. 28. Tomasz, A. 1969. Some aspects of the competent state in genetic transformation. Annu. Rev. Genet. 3:217-232. 29. Trautner, T. A., and H. Ch. Spatz. 1973. Transfection in Bacillus subtilis, p. 61-88. In W. Arber et al. (ed.), Current topics in microbiology and immunology, vol.

62. Springer-Verlag, Berlin. 30. Vermeulen, C. A., and G. Venema. 1974. Electron microscope and autoradiographic study of ultrastructural aspects of competence and deoxyribonucleic acid absorption in Bacillus subtilis: localization of uptake and transport of transforming deoxyribonucleic acid in competent cells. J. Bacteriol. 118:342-350. 31. Volkin, E. J. 1954. The linkage of glucose in coliphage nucleic acids. J. Am. Chem. Soc. 76:5892-5893. 32. Worcel, A., E. Burgi, J. Robinton, and C. L. Carlson. 1974. Studies on the folded chromosome of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol.

38:43-51.