Jan 13, 1975 - medium plus deoxyribonuclease I (100 ug/ml) and lysozyme (100 ug/ml) and incubated at 37 C for 40 min. The radioactivities were determined ...
JOURNAL OF BAcrERIOLOGY, Apr. 1975, p. 25-33 Copyright 0 1975 American Society for Microbiology
Vol. 122, No. 1 Printed in U.SA.
Different Nuclease Activities in Competent and Noncompetent Bacillus subtilis HANS JOENJE AND GERARD VENEMA* Department of Genetics, Biological Centre, University of Groningen, Kerklaan 30, Haren (Gn), T7he Netherlands Received for publication 13 January 1975
Competent and noncompetent cells of Bacillus subtilis were separated on the basis of their different buoyant densities. The two types of cells were compared with respect to their interactions with exogenous deoxyribonucleic acid (DNA). After exposure of DNA to the cells, the unadsorbed fraction of DNA molecules was examined. Both types of cells decreased the biological activity of this DNA, the inactivation exerted by noncompetent cells being more severe than that exerted by competent cells. Sedimentation analysis of the inactivated DNA revealed that fragments of DNA are produced, owing mainly to the introduction of doublestrand scissions. In addition to this fragmentation, the competent bacteria extensively digested the DNA exonucleolytically. This type of breakdown was specifically related to the competent state rather than to the state of low density. The exonucleolytic activity is, in all probability, associated with the cell envelope, because most of the activity is released into the medium when the cells are converted to protoplasts. At 37 C the competence-specific exonucleolytic breakdown started 2 to 3 min after the binding of DNA to the cells. In unfractionated cultures, breakdown may proceed until 70% of the total amount of DNA added has been made acid soluble. Nontransforming Escherichia coli DNA was also subject to exonucleolytic degradation; it seems unlikely, therefore, that this type of breakdown occurs as a consequence of recombination. Since ethylenediaminetetraacetate blocked both transformation by native DNA and the exonucleolytic breakdown of bound DNA, we suggest that the breakdown of DNA by competent cells fulfills an essential function in genetic transformation of B. subtilis.
Development of the physiological state of competence is a prerequisite for deoxyribonucleic acid (DNA)-mediated transformation in bacteria. In the case of Bacillus subtilis, maximally competent cultures always contain a majority (80 to 90%) of noncompetent cells (23). Although it has been established that competent cells differ in many respects from noncompetent cells (for a review, see reference 14), it is not clear which properties of a competent cell are essential for its transformability. In this paper we describe experiments aimed at characterizing properties of competent cells that are likely to be of significance to the process of transformation. To this purpose we separated competent and noncompetent cells and compared their interactions with exogenous DNA. Haseltine and Fox (9) have shown recently that exposure of DNA to noncompetent and competent B. subtilis causes inactivation of the DNA, the extent of inactivation being much more pronounced with noncompetent cells. These
observations have been qualitatively confirmed by our experiments. A novel observation made in our system is that competent cells have a powerful cell envelope-associated exonucleolytic activity, whereas noncompetent cells have much less of this activity. We suggest that this activity is essential for transformation of B. subtilis. MATERIALS AND METHODS
Strains. B. subtilis 1G-20 (trpC2) was used as the recipient in all transformations. Strain 1G-22 (thy) was used for the isolation of 3H-labeled transforming DNA. Unlabeled DNA was obtained from strain OG-1 (prototrophic). Media. For inoculation of overnight cultures, we used a minimal growth medium containing Spizizen (19) minimal salts, glucose (0.5%), casein hydrolysate (200 Ag/ml), and auxotrophic requirements (20 ug/ ml), except when stated otherwise. Cultures were grown to competence by diluting overnight cultures into WB medium containing Spizizen (19) minimal salts, glucose (0.5%), MgSO4 (6 mM), and compe25
26
J. BACTERIOL.
JOENJE AND VENEMA
tence-stimulating amino acids (50 Ag of each per ml), according to Wilson and Bott (25). DNA. Unlabeled and 3H-labeled transforming DNA were isolated from strains OG-1 and 1G-22, respectively, as described previously (10). "4C-labeled Escherichia coli DNA was isolated from a thyminerequiring E. coli strain by the same procedure, except that lysis of the cells was accomplished by 10 min of heating at 60 C in the presence of 2.5% sodium dodecyl sulfate. Maximally competent cultures. Maximally competent cultures (unfractionated) were obtained by using method B of Vermeulen and Venema (24). Separation of competent and noncompetent cells. Competent and noncompetent cells were separated by using a modification of the Cahn and Fox (2) and Hadden and Nester (8) centrifugation procedure, as described previously (11). Bacteria collected from the top, of the tubes after the Renografin centrifugation are highly competent and were designated as T bacteria. Noncompetent cells, collected from the bottom of the tubes, were designated as B bacteria. The transformability of T cells was usually 50- to 200-fold higher than that of B cells. Sucrose gradients. DNA was analyzed by sedimentation through linear sucrose gradients (5 to 20% in 0.15 M NaCl plus 0.015 M sodium citrate) using an SW50L swinging-bucket rotor in a Spinco L2 preparative ultracentrifuge (Beckman). Centrifugation was at 40,000 rpm and 20 C. Four-drop fractions were collected from the top, and their radioactivity was determined after addition of 0.25 ml of water and a suitable amount of scintillation fluid consisting of toluene plus Triton X-100 (10:3), and 5 g of 2,5diphenyloxazole and 0.05 g of 1,4-di-2-(5-phenyloxazolyl) benzene per liter of toluene. Molecular weights were calculated by using the equations of Burgi and Hershey (1), with an exponential constant of 0.35 for neutral gradients and 0.4 for alkaline gradients (20); T 7 DNA was used as a standard (double- and singlestrand molecular weights are 24.3 x 106 and 12.15 x 106 [22], respectively). Binding of DNA to cells as a function of time. At various intervals after the DNA and cells were mixed, the amount of cell-bound DNA was determined by adding 1-ml samples to 1 ml of ice-cold medium containing minimal salts (19), glucose (0.5%), calf thymus DNA (1 mg/ml), tryptophan (20 ,g/ml), and ethylenediaminetetraacetate (4 mM). Samples (1 ml) of these mixtures were layered on top of ice-cold stepwise sucrose gradients (6 ml of 5% sucrose on 8 ml of 10% sucrose in WB medium plus 20 mM ethylenediaminetetraacetate) and centrifuged for 15 min at 5,000 x g (2 C). After the tubes were decanted and dried, the pellets were suspended in minimal medium plus deoxyribonuclease I (100 ug/ml) and lysozyme (100 ug/ml) and incubated at 37 C for 40 min. The radioactivities were determined by adding 0.6-ml portions to suitable amounts of scintillation fluid. Assay of acid-soluble DNA breakdown products. Unless stated otherwise, 1-ml samples to be assayed were added to 1-ml portions of 6% perchloric acid, on ice. After standing at 0 C for at least 15 min, the precipitate was sedimented by centrifugation at
x g for 15 min, and 0.6 ml of the supernatant pipetted off for the determination of radioactiv-
10,000 was
ity.
RESULTS
Loss of transforming activity of DNA during exposure to T and B bacteria. The effect of exposure of DNA to T and B bacteria on the transforming activity of the unadsorbed DNA is shown in Fig. 1. Both types of bacteria inactivate transforming DNA, the B bacteria causing more extensive inactivation than the T bacteria: after 20 min of exposure the transforming activity had decreased to 6 and 17% of its initial value, respectively. Since transforming DNA is 0
2-
0
cn EL)
(n ca)
+0
v-
0~ _-
0 Ln
20 40 60 t i me (min)
FIG. 1. Loss of transforming activity of DNA during exposure to T and B bacteria. T bacteria at 1.8 x 108 (0) and B bacteria at 2.5 x 108 colony-forming units per ml (x) were incubated with 2 ,g of tritiated thy DNA per ml in WB medium at 37 C. After various intervals, samples to which ethylenediaminetetraacetate (40 mM) was added were removed. After centrifugation (10 min, 4,000 x g), the supernatant fluids were collected. The amounts of DNA equivalents present in the supernatants were calculated from their radioactivities. The transforming activity was determined by addition of 0.05-ml portions of the supernatant samples to 2-ml portions of a competent trpC2 culture, subsequent incubation at 37 C for 45 min, and selection for trpC2+ transformants. The number of transformants per microgram of DNA (specific transforming activity) was calculated for each sample. The values are plotted as percentages of the specific transforming activity of untreated DNA. The trpC2+ transformation frequencies of the T and B bacteria used in this experiment amounted to 2.1 and 0.023%, respectively. The specific transforming activity of the untreated DNA was 8 x 106 trpC2+ transformants per gg, assayed at a DNA concentration of 0.07 ,g/ml.
VOL. 122, 1975
27
COMPETENCE IN B. SUBTILIS
not inactivated when exposed to the culture fluids of either type of bacteria (data not shown), the inactivation observed requires a transient interaction of DNA with the cells. Nucleolytic activities associated with T and B cells. To examine whether the observed inactivation of DNA was due to nucleolytic attack we analyzed the unadsorbed radioactivity after 2.5, 15, and 40 min of exposure to T and B bacteria by centrifugation on sucrose gradients. During the 40-min exposure to B bacteria, the double-strand molecular weight of the DNA decreased from 15.8 x 106 to 3.3 x 106 (Table 1). When exposed to T bacteria, part of the DNA molecules became similarly fragmented but, in addition, an extensive degradation of DNA to very slowly sedimenting material occurred (Fig. 2). We conclude that during the transient interaction with T bacteria the DNA is subject to a severe exonucleolytic attack in addition to endonucleolytic degradation, whereas during such interaction with B bacteria degradation is restricted to endonucleolytic attack only. Comparison of the sedimentation profiles of DNA samples that had been exposed to T and B bacteria on neutral and alkaline sucrose gradients revealed that the observed fragmentation was due mainly to double-strand scissions (Table 1). Only a few additional single-strand breaks are introduced: about one single-strand nick in addition to each double-strand scission. The numbers of double- and single-strand breaks in DNA exposed to T and B cells are not essentially different. Since neither endonuclease nor exonuclease activity was detectable in the supernatant fluids of the cells (data not shown), the enzymes must be bound to the cells. The acid-soluble products generated from labeled DNA by T bacteria were mainly found in the culture fluid; after centrifugation of early samples, only a small fraction of these products (about 10%) was associated with the cells. The composition of the acid-soluble material in the culture fluid appeared to be different from that present in the cell pellet (Table 2). The cellassociated, labeled acid-soluble products were mainly thymidylic acid and its derivatives; only 2% oligonucleotides were present. In contrast, a considerable fraction of the products recovered from the culture fluid consisted of oligonucleotides, whereas the monomers were present mainly in the form of thymine. Relation between low density, transformability, and nuclease activity of cells. Since competent and noncompetent cells are separated on the basis of their different densities, properties of the T bacteria may relate either to competence or to their low density. To establish
TAsB
1. Double-strand (DS) and single-strand (SS) molecular weights of DNA exposed to Tand B bacteria
Duration of exposure
DNA mol wt
SS
DS o 2.5 15 40
B bacteria
T bacteriaa
15.8 x 12.6 x 8.3 x 3.3 x
106 10"
106
10'
4.5 3.0 1.5 0.7
x x x x
DS 10'
10' 10 106
15.8 x 12.6 x 5.4 x 2.3 x
SS
10' 10' 10'
10'
4.5 2.3 1.1 0.4
x x x x
106 10' 10' 106
a Only the peaks of highly polymerized DNA (see Fig. 2) have been used for these calculations.
whether the T cell-associated exonuclease activity was related to competence or to the state of low density, we determined during the growth of a culture: (i) competence, (ii) the fraction of cells having a low density, and (iii) the capacity of the cells to convert DNA into acid-soluble products as a measure for their exonuclease activity. The exonucleolytic DNA degrading capacity strictly paralleled the competence development of a culture (Fig. 3). The variation in the fraction of low-density cells followed a completely different pattern. This indicates that exhibition of exonucleolytic breakdown is specifically related to the competent state. Figure 3 also illustrates that attempts to separate competent and noncompetent cells by the method described are successful only in a certain growth phase of the cultures, namely at 0.5 to 1 h before maximal competence has been reached: then the fraction of competent cells equals that of cells having a low density (cf. reference 23), while simultaneously practically all of the competent cells are found in the low-density fraction. Location of exonuclease activity. In an attempt to locate the exonucleolytic activity, we converted cells into protoplasts with lysozyme and compared the enzyme activities exhibited by whole cells, the unfractionated protoplast suspension, the protoplast supernatant fluid, and the washed protoplasts (Table 3). After the T cells were converted to protoplasts, the enzyme activity decreased considerably; this was not expected since nothing had been removed from the suspension. Our explanation for this phenomenon is that the basic lysozyme molecules associate with the DNA, thus inhibiting its breakdown, because a similar inhibitory effect was observed when other basic polypeptides like spermine (Table 3), cytochrome c, or protamine were added at high concentrations to the cell-DNA mixtures (cf. reference 4). Furthermore, when lysozyme and DNA were added
J . BACTrERIOL .
JOENJE AND VENEMA
28
v
-
I~-I''
E 5 a,. T-bacteriaT
-,-_
T_III
II
CD4
TOP
10
TOP
TOP
10
10
fraction number FIG. 2. Sucrose gradient sedimentation patterns of DNA after exposure to T and B bacteria. Portions (2 ml) of T bacteria (2.8 x 108 colony-forming units per ml) and B bacteria (3.3 x 108 colony-forming units per ml) were incubated with 2 ,g of tritiated DNA at 37 C. After 2.5, 15, and 40 min, ethylenediaminetetraacetate (20 mM) was added, and the samples were chilled on ice. The bacteria were removed by layering the samples on 15% sucrose in a solution of 0.1 M potassium phosphate (pH 7.0) and 10 mM ethylenediaminetetraacetate and subsequent centrifugation in a swinging-bucket rotor (6,000 x g, 10 min, 2 C). The cell-free supernatant fluids were collected, and 0.1-ml portions were analyzed on linear neutral sucrose gradients as described. Upper gradients; DNA exposed to B bacteria. Lower gradients; DNA exposed to T bacteria. The arrows indicate the expected positions of T7 DNA (molecular weight, 24.3 x 106l) as determined from separate runs with T7 DNA. The direction of sedimentation is from left to the right. TABLE 2. Composition of acid-soluble DNA degradation products generated by T bacteriaa Recovered from:
Acid-soluble material (% of total input)
Supernatant Cells
26 2.7
% of radioactivity in:b Thymidine Oligonucleotides Thymidylic acid 39 2
7
81
3 3
Thymine 51 14
a 7.1 x 108 T bacteria per ml were incubated with 1 ,ug of 3H-labeled DNA per ml at 37 C for 9 min. After addition of 20 mM ethylenediaminetetraacetate, the suspension was chilled on ice and centrifuged through a discontinuous sucrose gradient (2 C). Acid-soluble material was obtained from the supernatant fluid and the pellet after trichloroacetic acid (final concentration 5%) was added; the solution stood for 2 h at 0 C, and the fluid was filtered. bAcid-soluble products were fractionated by gel filtration on Sephadex G-15 as described previously (11), except that the products were eluted with 5 mM sodium citrate, pH 10.4.
simultaneously, the same decrease of the nuclease activity was observed as when DNA was added after formation of the protoplasts. When the T cell protoplasts were separated from their supernatant fluid, most of the enzyme activity was found in the supernatant fraction. Only little activity was measured in the protoplast fraction. Removal of the protoplasts by centrifugation increased the activity in the supernatant to some extent. This may indicate that the protoplasts have an inhibitory effect on the enzyme activity present in the supernatant; the nature of this phenomenon was not further investigated, however. We conclude from these results that the exonucleolytic enzyme activity
is either located outside the cytoplasmic membrane or is loosely attached to it. Contrary to what was observed in T cells, B bacteria, having a low exonuclease activity, did not show a decreased nuclease activity when protoplasted with lysozyme (Table 3). Furthermore, spermine or lysozyme, added simultaneously with the DNA, did not seem to inhibit the low nuclease activity of B bacteria. The different sensitivity of the nuclease activity in B cells to these polycations might indicate that this activity is due to an enzyme different from that present in T cells. In contrast to what was found in T bacteria, after removal of the B protoplasts from their supernatant about equal activities
COMPETENCE IN B. SUBTILIS
VOL. 122, 1975
29
I
0
6 I
1-t
-12 -8-
£ a
0
40
-2-6 0 -6 o E
as-4
40
o
X
-2°
0o
Otime (h)
FIG. 3. Transformability (-), percentage of T bacteria (A), and DNA degrading capacity (0) as a function of growth in WB medium A trpC2 overnight culture in Pennassay broth (antibiotic medium III, Difco Laboratories) was suspended in WB medium to an absorbance at 450 nm (A4,0) of 0.27 and aerated at 37 C. Growth of the culture was followed by measuring the A4.. (0). After various intervals, samples were removed to determine the frequency of T bacteria after Renografin fractionation as described (A). Transformability (0) was measured by shaking 1-mI samples with trpC2+ DNA for 30 min at 37 C, followed by treatment with pancreatic deoxyribonuclease I (5 min, 20 ug/ml) and subsequent plating. The culture's DNA degrading capacity was measured by incubating 1-ml samples, in which the A4,, was adjusted to 3.0 (by concentration or dilution), with 1.7 ,g of tritiated DNA; after 40 min, trichloroacetic acid was added to a final concentration of 5% (wt/vol); after standing at 0 C for 2 h, the acid-precipitable products were collected by filtration through Whatman GF/C glass-fiber filters. The radioactivity of the acid-soluble products in the filtrates was measured and plotted as percentages of total DNA added.
were found in the protoplast fraction and in their supernatant; furthermore, removal of the B protoplasts did not lead to an enhancement of nuclease activity in the supernatant. We conclude that competent cells possess a surface-associated exonucleolytic activity that is most probably lacking in noncompetent cells. Binding and breakdown of DNA during exposure to competent cultures. Since the exonuclease activity described is predominantly exhibited by competent cells, it is conceivable that it is requisite to transformation. We investigated this possibility by testing the effect of ethylenediaminetetraacetate addition on the binding and breakdown of DNA. For this experiment, and also for the experiment presented in Fig. 5, we used unfractionated competent cultures, because both binding of DNA and breakdown to acid-soluble products do not occur in noncompetent cells. Figure 4 shows that the breakdown of DNA to acid-soluble products and transformation (see legend of Fig. 4) were blocked in the presence of ethylenediaminetetraacetate, whereas the binding of DNA was only moderately (40%) inhibited. This supports
the hypothesis that the competence-specific breakdown system is required for transformation of B. subtilis. Figure 4 also shows that binding of DNA started immediately after its addition (zero time), whereas the rapid production of acid-soluble material started after a lag period of 2 to 3 min; this breakdown continued for at least 60 min. The presence of this lag period indicates that binding of DNA precedes its degradation. The figure also shows that degradation continued after the net increase of DNA binding had stopped (after 15 to 20 min). This implies that after 15 min the DNA molecules continued to adsorb to the cells; however, the degradation and release of the DNA were apparently so extensive that no net increase of bound high-molecular-weight DNA was observed. We also examined the question of whether the exonucleolytic attack is specific for adsorbed transforming DNA. The data (Fig. 5) show that nontransforming E. coli DNA was also degraded, although this DNA is incorporated in a deoxyribonuclease-resistant state in B. subtilis (M. Piechowska, personal communication) without being genetically integrated (6). This
JOENJE AND VENEMA
30
J. BACTERIOL.
TABLE 3. Location of exonuclease activity
generated by exonucleolytic activity. At first sight it seems remarkable that exposure to noncompetent cells, only showing fragmentation of the DNA, causes a more severe loss of After pro- Protoplast toplastingb super- Protoplasts biological activity of the DNA than exposure to natant (total) the competent bacteria, which in addition degrade the DNA exonucleolytically. Haseltine 2 20 27 and Fox (9) have shown that, apart from 4 2 2 reducing the molecular weight, noncompetent cells inactivate the DNA in still another way, 22 3 15 4 1 2 manifesting itself in loss of capacity to compete Exonuclease activitya
Expt
1
2
Type of
Whole
cells
cellsb
Tc
59 (14)d
Bc
3 (5)d
Te
64 (7) 3 (2)t
Be
a The samples to be assayed were incubated with tritiated DNA (1.7 gg/ml) at 37 C for 30 min, trichloroacetic acid was added, and the amount of acid-soluble products was determined. The figures represent the amounts of acid-soluble DNA breakdown products as percentages of the total amount of DNA added. bWhole cells, suspended in a solution of 0.05 M potassium phosphate (pH 7.5), 10 mM MgSO4, and 20% sucrose (about 5 x 108 colony-forming units per ml), were either assayed directly for nuclease activity or after conversion into protoplasts with lysozyme (300 gg/ml at 37 C). Protoplast formation was checked by light microscopy. CThe trpC2+ transformation frequencies (1 ,ug of DNA/ml, 30 min, 37 C) amounted to 2.1% (T cells) and 0.02% (B cells). d Numbers in parentheses indicate activity when lysozyme and tritiated DNA were added simultaneously. e trpC2+ transformation frequencies amounted to 2.4% (T cells) and 0.04% (B cells). ' Numbers in parentheses are values obtained when spermine (3 mg/ml) was present in the incubation mixtures.
indicates that the exonucleolytic degradation is not due to a process intimately connected with
recombination.
0
20 40 time (min)
60
FIG. 4. Effect of ethylenediaminetetraacetate on
DISCUSSION binding and breakdown of DNA. A trpC2 competent When DNA is separately exposed to compe- culture was divided into two parts; to one part tent and noncompetent cells, the transforming ethylenediaminetetraacetate was added to a final of 15 mM. At zero time, 1.0 Ag of activity of the unadsorbed DNA decreases, the concentration 3H-labeled B. subtilis DNA was added to both parts, noncompetent fraction causing the most exten- and acid-soluble products and cell-bound DNA were sive inactivation. Haseltine and Fox (9) have determined in both cultures as a function of incubaobserved a similar phenomenon; however, they tion time at 37 C. Symbols: (0) binding, minus found less extensive inactivation by the compe- ethylenediaminetetraacetate; (A) binding, plus ethylenediaminetetraacetate; (0) breakdown, minus tent cell fraction than we did. Sedimentation analysis on sucrose gradients ethylenediaminetetraacetate; (A) breakdown, plus revealed that exposing DNA to the bacteria ethylenediaminetetraacetate. The number of transresults in its degradation. Both types of bacteria formants produced in both cultures was determined 30 min by 100-fold dilution of samples into fragmented the DNA to about the same extent, after minimal medium containing deoxyribonuclease I (1 mainly due to double-strand scissions, the mg/ml) and MgSO4 (10 mM), and subsequent plating competent cells showing, in addition, an ex- for trpC2+ transformants. The transformation fretensive breakdown to acid-soluble products. quencies amounted to 2.1% (without ethylenediThese products consist of more than 60% mono- aminetetraacetate) and 0.012% (with ethylenedimers (Table 2), so that they are most probably aminetetraacetate).
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COMPETENCE IN B. SUBTILIS
in transformation. This phenomenon has also been observed in our system (unpublished data) and explains the more severe inactivation of transforming DNA by noncompetent cells as compared to that by competent cells. The most significant difference observed between competent and noncompetent B. subtilis is that only competent cells show a powerful exonucleolytic breakdown, which ultimately may cause degradation of DNA to acid-soluble products up to 70% of the input (Fig. 5). These products are found predominantly in the culture fluid; after centrifugation of early samples, only a small fraction (10%) of the acid-soluble material may be found in the cell pellet. The cell-associated material and the released material appear to have different compositions (Table 2). The cell-associated, labeled products are mainly monomers, predominantly present as thymidylic acid; only a small fraction (2%) consists of oligomers (cf. 6). In contrast, the material recovered from the culture fluids consists of a considerable fraction of oligomers; the monomers are present mainly in the form of thymine. A possible explanation for the different compositions of the released and the cell-associated acid-soluble material might be a preferential release of oligomers and thymine as compared to the release of thymidylic acid. We cannot exclude, however, that the cellassociated and free acid-soluble materials are produced by different DNA breakdown systems generating different breakdown products. Basic proteins (lysozyme, cytochrome c, protamine, and spermine) strongly inhibit the exonucleolytic breakdown in competent cells. A similar effect of these proteins on DNA breakdown has been described by Davidoff-Abelson and Dubnau (4), who demonstrated that highmolecular-weight single-stranded donor DNA in lysates of newly transformed B. subtilis is recovered only when the cells are lysed in the presence of high concentrations of one of these basic proteins, which protect the DNA against nuclease(s). After forming protoplasts from the competent cells with lysozyme, we measured a decreased exonuclease activity; it seems likely that this is due to a protecting effect on DNA of the lysozyme against this nucleolytic activity. After centrifugation of the protoplasts, little activity is found in the protoplast fraction; most of the activity is measured in the supernatant. We concluded that the exonuclease is either located outside the cytoplasmic membrane or is loosely attached to it; it is possible that the enzyme is actually associated with the cellular mesosomes, which are expelled during protoplast formation (7, 16). The observation that the
31
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0
0.3-
W,°
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)'
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I
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_-
.,
,,/
.2
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.0, In I
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1
60 90 120 time (min) FIG. 5. Binding and breakdown of homologous and heterologous DNA. At zero time, a mixture of 14Clabeled E. coli DNA (39,000 counts/min per ig) and 3H-labeled B. subtilis DNA (150,000 counts/min per jig) was added to a competent culture (both DNAs at 0.44 jig/mI). After various intervals, samples were removed and acid-soluble breakdown products and cell-bound DNA were determined. Symbols: 0, E. coli DNA; 0, B. subtilis DNA; , cell-bound DNA; - - -, acid-soluble DNA. 0
30
protoplast suspension has a lower activity than the supernatant fluid may indicate that the protoplasts from competent cells contain an inhibitor to the nuclease; this inhibitory effect is not observed with protoplasts from noncompetent cells. The low level of exonuclease activity measured in noncompetent cells is not further decreased by lysozyme treatment of these cells, but rather seems to be slightly increased (Table 3). This may suggest that the actual exonuclease activity present in whole noncompetent cells is higher than the activity measured, but masked owing to the inability of these cells to adsorb DNA. This, however, would not agree with the observation that spermine, which has a strong inhibiting effect on nuclease activity in competent cells, has only a slight inhibiting effect in noncompetent cells. In view of these observations, it is conceivable that the low exonuclease activity in noncompetent cells is due to a nucleolytic activity different from that present in competent cells. Seto and Tomasz (17) have shown that, under certain conditions, induction of competence in pneumococci is accompanied by transport (or leakage) of exonuclease to the outside of the cytoplasmic membrane. Our results suggest
32
J.- BACTERIOL.
JOENJE AND VENEMA
that during the attainment of the competent state in B. subtilis a similar process may occur. The exonucleolytic breakdown system starts to attack the donor DNA 2 to 3 min after its adsorption to the cells (Fig. 4). About the same period of time elapses before the first recombinant DNA molecules are formed (5). Since only relatively small single-stranded pieces of adsorbed donor DNA are integrated, it should be questioned whether integration could account for the extent of breakdown observed. Two lines of evidence seem to exclude this possibility. Firstly, the observation that E. coli DNA, which is not genetically integrated in B. subtilis (6), is degraded similarly to transforming DNA (Fig. 5). Secondly, the fraction of irreversibly bound (deoxyribonuclease I resistant) DNA that is finally integrated amounts to approximately 20% (23). According to frequent observations in this laboratory, the absolute amount of DNA that is finally integrated amounts to about 0.01 ,ug of donor DNA per ml of competent culture exposed to transforming DNA at 1 ,g/ml. The amount of irreversibly absorbed DNA that can be rendered acid-soluble can, therefore, not exceed 0.05 ug of DNA equivalents per ml. Instead, we may find as much as 0.70 ,ug of acid-soluble products per ml. This indicates that the number of DNA molecules interacting with competent cells vastly exceeds that giving rise to transformants. Thus, it seems as if only a minor fraction of DNA escapes from total degradation and is integrated or, conversely, this DNA escapes destruction because of its being integrated. The location of the responsible nucleolytic activity (Table 3) suggests that breakdown is occurring outside or in association with the cellular membrane. A further inference is that the majority of the breakdown products is generated from reversibly bound (deoxyribonuclease I sensitive) DNA, because breakdown of irreversibly bound DNA can account only for a very minor fraction of the total amount of breakdown products generated. The observations that the exonucleolytic DNA degradation is exhibited by competent cells only and that ethylenediaminetetraacetate simultaneously inhibits this degradation system and transformation without drastically affecting the binding of DNA suggest that this breakdown system is involved in the transformation process. DNA molecules that become associated with competent cells in the presence of ethylenediaminetetraacetate are fixed in a state in which they are still accessible to externally added deoxyribonuclease I (13). This indicates that the conversion of bound DNA into a deoxyribonuclease-resistant state is a Mg2+_
dependent step. Results of others suggest (5. 15) that this step involves the conversion of doublestranded bound DNA into a single-stranded intermediate. It is conceivable that the competence-specific exonuclease described in this paper is the actual enzyme required for the irreversible fixation of bound donor DNA by converting double-stranded into single-stranded DNA fragments. A finding of others (3, 21), that addition of ethylenediaminetetraacetate blocks transformation mediated by native DNA but permits transformation with single-stranded DNA, supports this idea. Some similarities seem to exist between the transformation systems of B. subtilis and Diplococcus pneumoniae. In the latter organism, Seto and Tomasz (18) also observed that addition of ethylenediaminetetraacetate inhibits transformation and nuclease activity without inhibition of DNA binding. Lacks and co-workers (12) have recently characterized nontransformable D. pneumoniae mutants that are deficient in a particular nuclease; these mutants are able to bind DNA but unable to incorporate the bound DNA in a deoxyribonuclease I-resistant form. They conclude that the nuclease is probably involved in the irreversible incorporation of donor DNA. The present knowledge of the B. subtilis transformation system suggests that in this organism a similar mechanism of DNA incorporation may exist (also). ACKNOWLEDGMENTS We thank S. Bron, J. Buitenwerf, J. Kooistra, and M. Piechowska for valuable discussions, W. J. Feenstra for critically reading the manuscript, and L. Steendam for technical assistance during part of this research. This work was carried out under the auspices of The Netherlands Foundation for Chemical Research (S. 0. N.) and with financial aid from The Netherlands Organization for the Advancement of Pure Research (Z. W. 0.). LITERATURE CITED 1. Burgi, E., and A. D. Hershey. 1963. Sedimentation rate as a measure of molecular weight of DNA. Biophys. J.
3:309-321. 2. Cahn, F. H., and M. S. Fox. 1968. Fractionation of transformable bacteria from competent cultures of Bacillus subtilis on Renografin gradients. J. Bacteriol. 95:867-875. 3. Chilton, M. D., and B. Hall. 1968: Transforming activity in single-stranded DNA from Bacillus subtilis. J. Mol. Biol. 32:437-452. 4. 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 of donor origin. J. Bacteriol.
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