May 5, 1981 - solution contained about 250 ,ug of DNA per ml. A 20- ml sample of this solution was treated with acetic anhydride while it ... Bacterial strains used. Strain ... was incubated for 0.5 min at 37°C. The radioactivity bound by the ...
Vol. 149, No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1982, p. 595-605 0021-9193/82/020595-11$02.00/0
Uptake and Fate of Bacteriophage 4W-14 DNA in Competent Bacillus subtilis PALOMA LOPEZ,' M. ESPINOSA,' MIROSLAWA PIECHOWSKA,2* D.
SHUGAR,2 AND R. A. J.
WARREN3
Instituto de Inmunologia y Biologia Microbiana, Consejo Superior de Investigaciones Cientificas, Velazquez 144, Madrid-6, Spain'; Institute of Biochemistry and Biophysics, Academy of Sciences, 02-532 Warsaw, Poland2; and Department of Microbiology, University of British Columbia, Vancouver, Canada V6T I W53
Received t5 May 1981/Accepted 27 September 1981
Phage 4W-14 DNA (in which one-half of the thymine residues are replaced by a-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of 4W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of 4W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that 4OW-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of 4W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of fW-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of XW14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded 4W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in 4fW-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.
During the course of some investigations on the specificity of uptake by competent Bacillus subtilis of a senes of heterologous DNAs containing modified base moieties (11-14, 17, 18), our attention was drawn particularly to the behavior of DNA from phage (W-14, the host of which is Pseudomonas acidovorans; this phage neither infects nor transfects B. subtilis. The unusual composition of 4W-14 DNA, in which one-half of the thymine residues are replaced by 5-(4-aminobutylaminomethyl)uracil or a-putrescinyl thymine (7), is reflected by the unusual behavior of this heterologous DNA as a competitor in transformation and transfection systems (11-13). Two characteristics of 4)W-14 DNA are its more extensive binding by competent B. subtilis cells and the failure of an excess of homologous transforming DNA to reverse inhibition of transformation by this DNA (11). These observations pointed to the apparent existence of receptors for 4)W-14 DNA which differed from receptors for homologous DNA and the other types of heterologous DNA examined. In
this investigation we attempted to characterize further the nature and mechanism of uptake of (W-14 DNA.
MATERIALS AND METHODS Bacteral strai. The strains used are shown in Table 1. Preparation of DNA samples. The preparation of the various DNAs used has been described previously; these DNAs included B. subtilis 168' transforming DNA (17), labeled DNA from B. subtilis 168 thy (17), PBS1 DNA (11), and unlabeled (10) and labeled (6) 4)W-14 DNA. The molecular weights and specific activities of these DNAs were measured as described previously (18); values are given below where appropriate. Acetylation of +W-14 DNA. A solution of OW-14 DNA in 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) was dialyzed exhaustively against 0.1 M triethanolamine hydrochloride (pH 8.5). The final solution contained about 250 ,ug of DNA per ml. A 20ml sample of this solution was treated with acetic anhydride while it was stirred at room temperature. The pH of the solution was monitored during the reaction and was maintained at 8.5 by manually adding
595
5%
LOPEZ ET AL.
Strain B. subtilis 168 B. subtis 168 B. subtilis 168 B. subtilis 168 TKJ 6901 M. luteus ATCC 4698 E. coli K704 P. aeruginosa OT500 P. acidovorans ATCC 9355 P. acidovorans 6U
J. BACTERIOL.
TABLE 1. Bacterial strains used Genotype Prototroph thy trp thy thyA thyB urg-l Prototroph Prototroph Prototroph Prototroph ura
4 N NaOH. The acetic anhydride was added in five 0.1-ml portions, with intervals of 30 min between additions. The amount of acetic anhydride added (5.3 mmol) was 2.6 x 103 times more than the putrescinyl groups in the DNA sample. The solution was then dialyzed exhaustively against lx SSC. All of the primary amino groups in the putrescinyl groups were acetylated, as determined by the reaction of the DNA with trinitrobenzenesulfonic acid before and after acetylation (Warren, unpublished data). Competent cultures. Competent cultures were prepared (2) at densities of 1 x 108 to 2 x to 108 cells per ml, with competence in the range from 1 x 105 to 8 x 105 trp+ transformants per ml at a saturating concentration of 1 ,g of transforming DNA per ml. DNA uptake and competition. DNA uptake and competition were measured as previously reported (11). To 1.9 ml of a competent culture we added 0.1 ml of [3H]DNA at the requisite concentration, and the culture was incubated with shaking for 15 min at 37°C. Uptake was terminated by adding DNase I to a concentration of 20 ,ug/ml and incubating the preparation for 0.5 min at 37°C. The cells were centrifuged, washed, and precipitated with trichloroacetic acid (TCA). Uptake values were corrected for the radioactivity bound by a control containing 0.02 M MgCl2 that was added to a solution containing [3H]DNA and DNase I in 0.015 M NaCI-0.0015 M sodium citrate and was incubated for 0.5 min at 37°C. The radioactivity bound by the control culture was equal to 2% of that bound by the recipient culture after 15 min of incubation with native DNA at 37°C. Almost the same results were obtained when the control culture was incubated for 15 min with [3H]DNA previously degraded with DNase I (30-min digestion at 37°C). Potentially competing nonlabeled DNA was mixed with labeled DNA before it was added to the recipient culture; subsequent steps were as described above for measurements of DNA uptake. Any modifications from the standard procedures are referred to below in individual experiments. Cell lysates. Cells were lysed by treating them with lysozyme and Sarkosyl NL, followed by digestion with pronase, as described previously (17), except that the NaCl concentration during heating with Sarkosyl at 70°C was 0.15 M. Fractionation of DNA. DNA was fractionated according to density by centrifugation in a CsCl gradient at pH 11.2 (16, 17). The denatured 4W-14 DNA used as a density marker was obtained by heating 0.1-ml samples (2.5 ,ug in 0.25 M Tris-hydrochloride, pH 7.5,
Source
F. Makino G. Venema G. Venema A. A. Mercer R. Y. Stanier ATCC 9355
made 40%o in formamide) for 6 min at 80°C, followed by rapid cooling in an ice bath. Fractionation of competent cultures. Discontinuous gradients were used to fractionate competent cultures (2). Each of these gradients consisted of 0.5 ml of Urografin (76% aqueous solution of methylglucamineN,N'-diacetyl-3,5-diamino-2,4,6-triiodobenzoate) on the bottom as a cushion, 4 ml of Urografin diluted to a refractive index (no20) of 1.3713 as the separating layer, and 0.5 ml of a recipient culture prepared as follows. A suspension of competent cells was concentrated 10-fold by centrifugation and suspension of the cells in 0.1 volume of the supernatant; this suspension was kept at 37°C for 5 min and then incubated with DNA at 37°C for 20 min. After DNase was added, the suspension was incubated for an additional 5 min at 37°C. The cells that were concentrated at the top and bottom interfaces of the diluted separating layers were collected, centrifuged, and washed on a membrane filter (type HAWP; 0.45 ,um; Millipore Corp., Bedford, Mass.) with LS growth medium (2), then with 0.2 M MgC92, and again with LS medium. The cells from the top fraction were suspended in 0.5 ml of growth medium 2 (2), and those from the bottom fraction were suspended in 2.5 ml. These preparations were monitored for the number of viable cells, the number of trp+ transformants, and radioactive DNA uptake. Chemicals. Sodium azide was obtained from E. Merck AG, Darmstadt, Germany, and was prepared as an aqueous solution immediately before use. Urografin was from Schering Espana S.A., Madrid, Spain. Chloramphenicol, as the sodium succinate salt (0.77 mg of chloramphenicol per mg), was a gift from Laboratories Normon S.A., Madrid, Spain.
RESULTS time dependence of DNA and Concentration uptake. Previous preliminary experiments pointed to more efficient uptake of 4)W-14 DNA than of B. subtilis DNA (11). More detailed studies of the concentration dependence of DNA uptake (Fig. 1) confirmed the earlier observations and showed that at varying subsaturating DNA concentrations, the amount of OW-14 DNA taken up was threefold higher than the amount of homologous DNA taken up. Saturation by 4W14 DNA was attained at a concentration of 0.5 gg/ml, compared with 1 ,ug/ml for B. subtilis DNA; however, the lower saturation concentra-
UPTAKE AND FATE OF OW-14 DNA
VOL. 149, 1982
CL
8 101
z
0.1
1
3H-DNA [jig/ml I
FIG. 1. Uptake of [3H]DNA by competent B. subtilis cells as a function of DNA concentration. Symbols: 0, 4W-14 [3H]DNA (specific activity, 5.5 x 104 cpm/,Lg; molecular weight, 107; radioactivity measured at a plateau of 2,500 cpm/sample); 0, B. subtilis [3H]DNA (specific activity 6.2 x 10 cpm/nug; molecular weight, 2 x 107; radioactivity taken up in the presence of 1 ,ug of DNA per ml 15,780 cpm/sample). Measurements were corrected for background counts at varying [3H]DNA concentrations.
tion for the former was probably due to the fact that its molecular weight was only one-half that of B. subtilis DNA (Fig. 1) (3). At saturating concentrations, the amount of 4W-14 DNA taken up was about twice the amount of B. subtilis DNA taken up and was equal to 1.2 x 10-2 ,ug/ 108 colony-forming units (CFU). In other terms, about seven molecules of 4W-14 DNA or two molecules of the higher-molecular-weight homologous DNA were taken up per CFU. Assuming that 20%o of the cells were competent and that only those cells took up DNA (2), it followed that about 35 molecules of 4~W-14 DNA or about 10 molecules of B. subtilis DNA were taken up per competent cell. An examination of the kinetics of DNA uptake (Fig. 2) also showed a higher rate of uptake for 4W-14 DNA. For the initial period of uptake (2.5 min), the rates were 14 x 10-4 ,ug of tW-14 DNA per 108 CFU per min and 3.4 x 10- ,ug of homologous DNA per 108 CFU per min, a fourfold difference. A portion of the TCA-precipitable donor DNA label taken up is gradually lost during subsequent incubation of cells (17). Therefore, the amount of DNA label measured as taken up was in fact the difference between the actual uptake and the loss of TCA-precipitable label. Comparisons of DNA retention (Fig. 3) showed that 4W14 DNA was degraded to TCA-soluble material after uptake at a faster rate than homologous DNA. However, degradation stopped after about 10 min of incubation, and 50% of the label remained TCA insoluble. Is +W-14 DNA taken up exclusively via ho-
597
mologous DNA receptors? This question arose as a consequence of the results of some of the competition experiments (11) referred to above. Additional experiments (Fig. 4) demonstrated that there was no substantial competition by homologous DNA for uptake of iW-14 DNA under conditions where cold 4W-14 DNA exhibited competition (Fig. 4A) and that labeled homologous DNA uptake was reduced to a normal level by either cold homologous DNA or cold XW-14 DNA (Fig. 4B). It should be noted that the accuracy of our DNA uptake measurements normally was about +4%, but occasionally was higher (e.g., an uncertainty of 13% is apparent in Fig. 4A). Furthermore, the effects shown in Fig. 4B were, reproducible with much lower concentrations of 4W-14 DNA, as described elsewhere (11).
Iv
0
152 10 50
Ti me of upta ke I[min ] FIG. 2. Kinefics of uptake of W-14 [3H]DNA (0) and B. subtilis [3H]DNA (O) by 1-ml samples of B. subtilis recipient cultures at a DNA concentration of 1 tLg/ml. The DNA samples used were the same as those described in the legend to Fig. 1 and Table 2.
598
LOPEZ ET AL.
80\ -
0
aa
60
-~
40
6
20
30 10 60 Time of incubation [ min] FIG. 3. Retention by competent B. subtilis cells of TCA-precipitable label of 4W-14 [3H]DNA (0) and B. subtilis [3H]DNA (0). Uptake was measured by using 1-ml samples of cells at a DNA concentration of 1 ,ug/ml. Retention was measured during incubation at 37°C after DNase was added. The 100% value for retention of VW-14 DNA label (specific activity, 6.6 x 104 cpm/,g) was 1,887 cpm after 0.5 min of incubation with DNase; for B. subtilis [3H]DNA (specific activity, 2.7 x 10- cpm/,lg) the corresponding value was 5,684 cpm.
A comparison of the kinetics of 4)W-14 [3H]DNA uptake in the absence and in the presence of cold B. subtilis DNA (added in a 1:1 ratio; final concentration, 1 pLg/ml each) showed no detectable influence of the B. subtilis DNA (Fig. 5A); the rate of uptake of 4W-14 DNA remained at the level of 14 x 10" ,g/108 CFU per min, as in the experiments shown in Fig. 2. By contrast, the rate of uptake of B. subtilis [3H]DNA was reduced about one-half (to 1.6 x 10-4 jLg/108 cells per min) by the addition of an equal concentration of (W-14 DNA (Fig. SB), as expected with a total DNA concentration of 2 p,g/ml, which was saturating for the recipient population. The rate of uptake of B. subtilis DNA alone (3.3 x 10-4 ,ug/108 CFU per min) was almost identical to the value obtained in previous experiments (Fig. 2). Poor competition for uptake of labeled 4)W-14 DNA was also observed with two other types of DNA, namely, acetylated 4W-14 DNA and phage PBS1 DNA (Table 2). The simultaneous addition of either of these potentially competing DNAs to a concentration of 5 pug/ml of recipient
J. BACTERIOL.
culture and of labeled 4W-14 DNA to a concentration of 1 ,ug/ml decreased uptake of the latter by only about 30%o, whereas addition of cold normal 4W-14 DNA reduced uptake by 70%, close to the value expected for dilution of labeled molecules. Normal competition for uptake of labeled homologous DNA was also observed in control experiments when unlabeled homologous DNA, 4W-14 DNA, acetylated 4W-14 DNA, and phage PBS1 DNA were used as competitors (Table 2). Potential competition was also examined under conditions that were more advantageous for binding of homologous DNA to cell receptors. B. subtilis DNA was added to a recipient culture to a concentration of 5 ,ug/ml at 2.5, 5, and 15 min before the addition of 4W-14 [3H]DNA to a concentration of 1 ,ug/ml. Even under such conditions, uptake of the latter was not reduced by more than 4 to 5%, which was within the range of experimental error (see above) and obviously was quite different from the 90%o decrease in uptake of B. subtilis [3H]DNA that occurred after the addition of cold B. subtilis DNA under the same experimental conditions (Table 3). Specificity of uptake of +W-14 DNA. The results presented above suggested that 4W-14 DNA penetrated B. subtilis cells not only via receptors for homologous molecules, but also via some other pathway (see below). It was of obvious relevance to establish whether uptake of +W-14 DNA was dependent on cell competence. Experiments performed with competent and noncompetent cultures of B. subtilis and with noncompetent cultures of Micrococcus luteus, Escherichia coli, and Pseudomonas aeruginosa demonstrated that uptake was limited to competent cultures of B. subtilis (Table 4). Obvi-
2
3
4
5
1
2
3
Competing DNA [lpg/mll FIG. 4. Competition for uptake of 4W-14 [3H]DNA (A) and B. subtilis [3H]DNA (B), each at a concentration of 1 ,ug/ml of recipient culture, by varying concentrations of cold +W-14 DNA (0) and B. subtilis DNA (0). The 100o values for uptake by 1-ml samples of recipient cultures in the absence of competing DNA were 1,280 cpm for 4~W-14 [3H]DNA and
3,571 cpm for B. subtilis [3H]DNA. Both the labeled and the cold DNA samples were the same as those described in the legend to Fig. 1 and Table 2.
UPTAKE AND FATE OF 4W-14 DNA
VOL . 149, 1982
0~~~~~~~~~
5
10
15
20
0
5
10
15
20
Time of DNA uptoke [min] FIG. 5. Kinetics of uptake of 4W-14 [3H]DNA in the presence (O) and in the absence (0) of cold B. subtilis DNA (A) and of B. subtilis [3H]DNA in the presence (O) and in the absence (0) of cold 4W-14 DNA (B). Labeled and competing DNAs were each added to a concentration of I ,ug/ml of competent culture. The experimental conditions were as described in the legends to Fig. 1 and 2 and in Table 2.
described above, the efficiency of 4W-14 DNA uptake was not altered by the presence of homologous DNA, whereas the efficiency of transformation was reduced about 100-fold by the presence of 4W-14 DNA. The facile detectability of trp+ transformants simplified monitoring of fractionation by allowing determination of the distribution of competent cells relative to the distribution of 4W-14 DNA label taken up. Fractionation led to concentration of both the transformants and the label in the top fraction (Table 5). The ratios of values in top fractions to values in bottom fractions were 800 and 900 for the 4W-14 DNA label and the trp+ transformants, respectively. The temperature dependence of uptake of 4~W-14 DNA was also similar to that of homologous DNA uptake (Table 6). Uptake decreased by one-half when the temperature was lowered from 37 to 30°C and fell to zero at 2°C. In the presence of 15 mM EDTA, uptake of 4W-14 DNA at 37°C decreased to 3% of the control value (Table 7); a similar decrease was observed for uptake of B. subtilis DNA. The efficiency of trp+ transformation was much more sensitive to EDTA, with a decrease of 4 TABLE 2. Competition by various DNAs for uptake of phage 4.W-14 or B. subtilis [3H]DNAa [3H]DNA (1 p.g/ml)
4W-14
Competing DNA Expt P (5 Lg/ml)
1 2
ously, it would be desirable to establish whether
other known competent strains take up (W-14 DNA. Since only 10 to 20% of the CFU in a B. subtilis competent population are actually competent (i.e., capable of taking up and expressing the traits of transforming DNA [2]), it was of DNA logical to inquire whether uptake4WW-14 was limited to the same fraction of the population. That this was indeed the case was shown by fractionation of a recipient culture after uptake of W-14 [3H]DNA or B. subtilis [3H]DNA (Table S). The amount of label per CFU was about 2efold higher in the 4% of the population that was isolated in the so-called top fraction of a Urografin density gradient. The same top fraction exhibited a 25-fold higher frequency of trp+ transformants (8.5%) than the nonfractionated population (0.36al). Even more unequivocal were the results of experiments in which a competent culture was N exposed tonuig ofwW-14 [3uH]DNA per ml and wag of B. subtilis DNA per ml, followed by fractionation on a Urografin gradient. In agreement with previous results (10) and the results
599
3 4
None B. subtilis None B. subtilis None B. subtilis PBS1 None
4W-14 Acetylated 4W-14 5
6
None 4W-14 None
,OW-14 B. subtilis
None
4~W-14
DNA uptake (cpm) 1,281 (100)b
1,257 1,790 1,786 1,136 1,075 816 1,256 391 859
(98)
(100) (100) (100) (95)
(72) (31)
(100)
(68)
1,220 368 1,734 525
(100)
4,927 890
(100) (18) (21)
(100)
(30) (30)
1,017 Acetylated 4W-14 B. subtilis 902 (18) PBS1 472 (10) a Competition experiments were performed as described in the text, except that 0.9 ml of a competent culture was used. Molecular weights and the specific activities of [3H]DNAs were determined as described in the legend to Fig. 1. The molecular weights of cold competing B. subtilis, PBS1, and 4W-14 DNAs were 1.3 x 107, 1.4 x 10', and 1.2 x 107, respectively. b The numbers in parentheses are percentages.
600
LOPEZ ET AL.
J. BACTERIOL.
with 20 mM azide (Table 7); cell viability
TABLE 3. Influence of presaturation of a B. subtilis competent culture with homologous DNA on the uptake of 4W-14 or B. subtilis [3H]DNAa Competing DNA % Uptake Time of addition or [3H]DNA
was
not affected by exposure to these concentrations
of azide. Bearing in mind that acquisition of competence by B. subtilis is sensitive to inhibitors of protein synthesis (19), it was pertinent to inquire Presence preincubation (min) whether this prevailed for the ability to take up -15 4W-14 100 4W-14 DNA. In fact, incubation of a recipient 0 4W-14 100 culture with chloramphenicol for 2.5 h before + -15 OW-14 95 the of the peak of competence led to appearance + -10 4W-14 101 a decrease in the uptake of 4W-14 or homolo+ -5 4W-14 9 gous DNA to about 20% of the control value. + -2.5 OW-14 97 Under these conditions, which did not affect cell + 0 4W-14 108 viability, the number of trp+ transformants was 0 B. subtilis 100 reduced 10-fold. + -15 B. subtilis 10 Fate of pW-14 DNA in recipient cells. Recipi+ -5 B. subtilis 10 ent cell lysates containing [14C]thymidine-laa At the times indicated, cold homologous DNA was beled DNA were fractionated on pH 11.2 CsCl added to 1-ml samples to a concentration of 5 ,ug/ml. gradients after uptake of 4W-14 [3H]DNA at Samples were brought to 37°C and incubated to zero 30°C. This lower uptake temperature was selecttime, and B. subtilis [3H]DNA (specific activity, 1.0 x ed to facilitate a comparison of the fate of 4W-14 105 cpm/,ug) or 4W-14 [3H]DNA (specific activity, 5.5 DNA with the fate of other types of DNA by x 104 cpnm/pg) was added to a concentration of 1 ,ug/ reducing the rate of recombination (16). Analyml. This was followed by incubation for 15 min at 37°C ses were based on determinations of the relative and appropriate treatment of samples for uptake mea- positions of recipient and donor DNAs in consurements. The 100%o value for uptake of 4W-14 [3H]DNA was 1,375 cpm, and the 100%o value for trol gradients of lysates of cells which were uptake of B. subtilis [3H]DNA was 1,110 cpm (in not treated with donor DNA and to which 4iW14 DNA was added just before centrifugation. absence of competing DNA). As expected, native 4W-14 DNA was found at a lower density than B. subtilis DNA, with a orders of magnitude; cell viability was not af- displacement from recipient DNA equal to 13% fected under these conditions. In the presence of of the length of the gradient (Fig. 6A). Dena10 and 20 mM sodium azide, an inhibitor of tured 4W-14 DNA was denser than B. subtilis oxidative phosphorylation, uptake of 4fW-14 DNA and was displaced from recipient DNA by DNA decreased to 0.7 and 0.4%, respectively, 5% of the length of the gradient in the opposite of the control value. A similar sensitivity of direction (Fig. 6B). About 12% of the native homologous DNA uptake to azide was ob- 4W-14 DNA added to a control mixture was served, together with a reduction in the number found in the recipient DNA band, most likely as a of trp+ transformants by 2 orders of magnitude result of trapping during centrifugation, although with 10 mM azide and 3 orders of magnitude the total amount of DNA in the gradient was TABLE 4. Uptake of phage 4W-14 or B. subtilis [3H]DNA by competent (i.e., transformable) and noncompetent 1-ml samples of bacterial cells Bacteria
Culture medium
B. subtilis 168 thy trp
Competent
GMII
Noncompetent
GMII Penassay broth
CFU/ml
[3HJDNA
Background(cpm) radioactivity
1.5 x 108
4W-14a B. subtilisb
82 36 47
8,455 1,100 40
28
43 52 43 29 66 58
9 X 107 6 x 107
107
M. luteus ATCC 4698
Penassay broth
4x
E. coli K704
Penassay broth
6 x 108
P. aeruginosa OT500
Penassay broth
Specific activity, 4.0 x 105 cpm/,Lg. b Specific activity, 1.0 x 105 cpm/,ug. c Specific activity, 6.6 x 104 cpm/Lg.
a
6x
108
OW-14C
4W-14a B. subtilisb 4W-14a B. subtilisb
4W-14a
B. subtilisb OW-14a B. subtilish
30 42 26 50 38 48 39
Uptake (cpm) Utk cm
65 53
VOL. 149, 1982
only about 2 ,ug. Similar trapping was found in the control with denatured 4W-14 DNA. This was probably due to the high molecular weight of the homologous DNA, and such effects have been observed previously (8, 17). Figures 6C and D show the results for a recipient lysate whose cells had been incubated at 37°C for 0.5 and 20 min, respectively, after the termination of 4W-14 DNA uptake. Most of the donor DNA label in the 0.5-min sample banded at the position that was characteristic of denatured DNA, and the remainder coincided with the recipient DNA band. The 20-min sample showed no denatured 4W-14 DNA. About onethird of the total donor label of the 0.5-min sample was still present in the 20-min sample, and this label was associated with the recipient DNA band. DISCUSSION The curves for the concentration and time dependence of 4W-14 DNA uptake (Fig. 1 and 2) clearly show that this DNA is taken up three times faster than homologous DNA, whereas the amount taken up is at least twice that of homologous DNA. This may be explained in the following ways: (i) more rapid uptake of VW-14 DNA via homologous DNA receptors; (ii) simultaneous uptake via homologous receptors and some other pathway; and (iii) slower degradation after uptake. The latter possibility must be considered since previous observations on the uptake and fate of heterologous DNA (17) demonstrated that the radiolabel of DNA taken up and found in cells in a TCA-precipitable form is the sum of the amount taken up and present in polymerized form, the fraction degraded within the cells after uptake, and the fraction incorporated into recipient DNA in the form of monomers. The results of this investigation exclude this possibility since they show the reverse effect (i.e., a twofold faster decrease in cellbound radiolabel of 4W-14 DNA compared with homologous DNA [Fig. 3]). The more rapid degradation of FW-14 DNA after uptake provides a reasonable interpretation for the difference between our findings and those reported previously (13), namely, a larger uptake of 4W-14 DNA with increasing subsaturating concentrations than observed here (Fig. 1). It had been observed previously that at a DNA concentration of 0.05 ,ug/ml (well below saturation), the amount of 4W-14 DNA taken up was eightfold higher than the amount of homologous DNA taken up, whereas the present experiments showed that the amount of XW-14 DNA taken up was only threefold higher (Fig. 1). It is now clear that the difference in the amounts of uptake decreased with an increase in the DNA concentration in the medium due to factor(s)
UPTAKE AND FATE OF OW-14 DNA
601
which reduced the relative extent of degradation of the DNA taken up (e.g., by inhibition or saturation of nucleolytic enzymes). The differences between the previous results (13) and the present results were most likely due to differences in the methods of measurement; in the earlier experiments the extent of uptake was based on measurements of the total radioactivity of the DNA taken up; in this study we measured the TCA-precipitable radioactivity. Consequently, the differences between the earlier results and the results of this study indicate that the products of degradation of 4W-14 DNA after uptake are retained in the cells in acid-soluble form and that the quantitative differences in the uptake of 4W-14 and B. subtilis DNAs shown in Fig. 1 and 2 are lower than the real values. It should be noted that measurement of acid-precipitable radioactivity has become more widely accepted and applied to monitor DNA uptake than measurements of total radioactivity bound by cells. In connection with the results discussed above, it is relevant that the levels of uptake of other types of heterologous DNA by competent B. subtilis cells, measured as TCA-precipitable material, are lower than the levels of uptake of homologous DNA, ranging from about 7% for phage T6 DNA to 37 to 65% for E. coli and about 90%o for non-glucosylated T6 DNA (18). The possibility of a more rapid uptake of 4W14 DNA via homologous DNA receptors was excluded by a comparison of the kinetics of uptake of homologous DNA in the absence and in the presence of 4W-14 DNA (Fig. 5). The twofold reduction in the rate of uptake of labeled B. subtilis DNA resulting from the addition of an equal quantity of cold competing 4W-14 DNA is what might be expected from dilution with homologous DNA (on a weight basis). In fact, the dilution of labeled homologous DNA molecules by cold 4W-14 DNA molecules was threefold, since the molecular weight of the latter was only one-half that of the former. The reduction in the rate of uptake of homologous DNA by only twofold suggests that the affinity of 4W-14 DNA for homologous DNA receptors is lower than that of homologous DNA. Hence, the fact that more 4W-14 DNA than homologous DNA is taken up must be due to more effective uptake via some pathway other than receptors for homologous DNA. Such a conclusion had been reached already on the basis of earlier observations (11, 13) and was reinforced by the absence of any competition for uptake of 4W-14 DNA by homologous DNA (Fig. 4 and 5A and Tables 2 and 3). Such presumed additional receptors are obviously related to competence, since noncompetent cells do not take up any DNA, including 4W-14 DNA. By contrast, the lower but experi-
602
LOPEZ ET AL.
J. BACTERIOL.
TABLE 5. Fractionation of competent cultures of B. subtilis on discontinuous Urografin gradients Before fractionation DNA uptake
No. of cells placed on gradient No. of trp' transformants
Source of [3H1DNA
Total no.
B. subtilis (5 ,ug/ml)
8.2 x 108
4~W-14 (5 ,ug/ml)
8.2 x 108
vW-14 (5 pg/ml) + cold B. subtilis (5 ,ug/ml)
8.2 x 108
N
Band
Frequency (%)
cpm
ILg/108 CFU
0.36
6,371
0.4 x 10-2
Top Bottom
12,211
1.0 x 10-2
Top Bottom
12,015
1.0 x 10-2
Top Bottom
3.0 x 106
2.1 x 104
0.003
The specific activities of B. subtilis and 4W-14 [3H]DNAs were 1.85 x 105 and 1.46 x 105 cpm, respectively. digested with DNase for 30 min at 37°C; background values were 41 cpm for the unfractionated culture, 38 cpm The corresponding values for a culture treated with degraded 4W-14 [3H]DNA were 39 and 13 cpm for the top a
mentally detectable (i.e., exceeding the 4% error in uptake measurements) competition by PBS1 DNA and acetylated 4OW-14 DNA for uptake of normal 4W-14 DNA (Table 2) indicates some affinity of the two former DNAs for the pathway of uptake of 4W-14 DNA, notwithstanding that these DNAs also exhibit normal competition for uptake of homologous DNA. It is significant that there is some evidence for the presence of two types of binding sites in competent gonococci (4). The nature of the sites in B. subtilis is being investigated further in our laboratories. Acetylation of 4W-14 DNA, which leads to a decrease in competition with nonmodified 4W14 DNA, indicated the significance of the charged putrescinyl amino groups in the transport of this DNA into cells. However, since
acetylation also increases the bulk of the putrescinyl residue, which is localized in the major groove of the DNA helix, it is difficult to conclude whether charge neutralization or enhanced steric effects or both are responsible for the decreased affinity for 4W-14 DNA receptors. In considering whether the mechanism of 4OW14 DNA uptake involves active transport via specific receptors, as for homologous DNA or other types of heterologous DNAs (15, 19), it should first be noted that the concentration dependence and time dependence of 4W-14 DNA uptake follow courses similar to those for homologous DNA and that uptake levels off at times and concentrations similar to those for homologous DNA (Fig. 1 and 2). Furthermore, fractionation of a competent population showed
TABLE 6. Influence of temperature on uptake by B. subtilis competent cells of phage 4W-14 or B. subtilis [3H]DNA Temp of uptake (QC 37
[3H]DNA 4W-14 B. subtilis
30
DNA taken upa
Sp act (cpm4Lg) 6.6 x 104 4.0 x 105 2.7 x 105 1.0 x 105
Source
6.6 4.0 2.7 1.0
4W-14 B. subtilis
104 105 x 10' x 105
x x
cpm/108 CFU 1,691 6,589 3,136 1,014 951
3,672 1,838 507
iLg/108 CFU 6.4 x 10-3b 6.1 x 10-3c 2.9 x 10-3b 3.6 x 10-3c
3.6 3.4 1.7 1.8
x 10-3b x 10-3c x 103b x 10-3c
UDd UD 4.0 x 10 OW-14 UD UD 2.7 x 105 B. subtilis a The ratio of the mean value for 4W-14 DNA to the mean value for B. subtilis DNA was 1.9 at 37°C and 2.1 at 2
300C. b Density of competent population, 4.0 c Density of competent population, 2.7 d UD, Undetectable.
x x
108 CFU/ml.
10" CFU/ml.
UPTAKE AND FATE OF 4W-14 DNA
VOL. 149, 1982
603
after uptake of phage +W-14 or B. subtilis [3HIDNAa After fractionation No. of cells collected No. of trp+ transformants
Total no. No.
3.3 x 107 7.1 x 108
2.8 x 106 8.2 x 104
Frequency 8.5 0.01
4 87
93 3
1.7 x 104 4.9 x 102
0.05 6 x 10-5
5 96
DNA uptake
81 2
Top/bottom ratio Trans-
cpm
~Lg/108 CFU
5,832 410
9.5 x 10-2 3.1 x 10-4
238
2.6 x 10-1 2.2 x 10-4
11,680
2.2 x 10-1 2.3 x 10-4
11,591
4 90
3.1 x 107 7.4 x 10" 3.7 x 107 7.9 x 108
% Recovery TransCells formats
260
formation
Uptake
frequency
(p.g/108 cpm)
848
306 1,182
833
956
DNA uptake was corrected for background, as measured in samples that were treated with [3H]DNA previously for the top fraction, and 14 cpm for the bottom fraction of a culture treated with degraded B. subtilis [3H]DNA. and bottom fractions, respectively.
that XW-14 DNA is taken up by the same fraction which takes up and is transformed by B. subtilis DNA (Table 5), which is consistent with the finding that neither DNA is taken up by
noncompetent cultures of B. subtilis or other organisms (Table 4). The EDTA-induced decrease in 4W-14 DNA uptake is similar to the decrease in homologous
TABLE 7. Influence of metabolic inhibitors and EDTA on the uptake of OW-14 or B. subtilis [3H]DNA by competent B. subtilis cells (3.4 x 10' CFU/ml) and on transformation' No. of [3H]DNA Inhibitor [3H]DNA Exptb transformantstrpe per ml CFU) up (p.g/1081 taken 1.1 x 106 (100)C 4.0 x 10-3 None B. subtilis 1 1.2 x 102 1.4 x 10-4 (4) EDTA
2
3
4W-14
None EDTA
1.0 x 10-2 2.6 x 10-4
(100) (3)
B. subtilis
None NaN3 (10 mM) NaN3 (20 mM)
3.9 x 10-3 7.5 x 10-5 2.9 x 10-5
(100) (2) (1)
4W-14
None NaN3 (10 mM) NaN3 (20 mM)
1.0 x 10-2 7.3 x 10-5 4.1 x 10-5
(100) (1) (0.5)
B. subtilis
None Chloramphenicol
4.0 x 10-3 7.9 x 10-4
(100) (20)
1.4 x 106 2.8 x 104 2.3 x 103
1.4 x 106 1.8 x 105
(100) 1.1 x 10-2 None (21) 2.3 x 10-3 Chloramphenicol a The specific activities of B. subtilis and 4W-14 DNAs were 1.85 x 105 and 1.46 x 105 cpm/4ig, respectively; the background values were 43 and 36 cpm, respectively. The maximal effect on cell viability was 3.5%. b In experiment 1 EDTA was added to a concentration of 15 mM, and competent cultures were incubated for 5 min at 37°C; this was followed by the addition of [3H]DNA and incubation for 15 min. A 1-ml sample was centrifuged, the resulting cell pellet was suspended in 1 ml of fresh medium containing 0.02 M MgCI2, DNase I was added to a concentration of 20 ,g/ml, and the preparation was incubated for 2 min to degrade extracellular DNA. Cells were again pelleted and washed, and the radioactivity of the donor DNA was measured. In experiment 2 NaN3 was added 10 min before the peak of competence was attained by the recipient culture at 30"C; this was followed by a 10-min incubation at 37°C and the subsequent addition of [3H]DNA for uptake measurements. In experiment 3 chloramphenicol was added to a concentration of 20 ,ug/ml of recipient culture 150 min before the peak of competence was attained and [3H]DNA was added. I The numbers in parentheses are percentages. 4W-14
604
LOPEZ ET AL.
u
J. BACTERIOL.
~~~CD
CL
3
30
50
30
50
30
50-
30
50
2
Fraction number FIG. 6. Fate of OW-14 DNA after uptake by B. subtilis recipient cells, based on a CsCl density gradient analysis of cell lysates. (A) Relative positions in a gradient of native 4W-14 DNA (A) and native B. subtilis recipient DNA from a cell lysate (A). (B) Relative positions of denatured W-14 DNA (A) and native B. subtilis DNA from a cell lysate (A). (C and D) Analysis of lysates of cells labeled with [14C]thymidine (A) and incubated for 0.5 and 20 min, respectively, after uptake of 4W-14 [3H]DNA (A) (specific activity 5.5 x 104 cpm/,lg) for 15 min at 30°C.
DNA uptake by competent B. subtilis and other strains (5, 15, 19) and suggests that there is a requirement for divalent cations in this process. In addition, 4W-14 DNA uptake exhibits the same dependence on temperature and on energy derived from oxidative phosphorylation as homologous DNA. The significance of the latter is emphasized by the azide-induced decrease in DNA uptake by B. subtilis (20) and Haemophilus iiifluenzae (1). In conclusion, 4OW-14 DNA uptake exhibits the characteristics of uptake by active transport of homologous DNA (9, 15, 19). This similarity also extends to the dependence of competence development on protein synthesis, as illustrated by the chloramphenicol-induced decreases in transformation (Table 6) (15, 19) and in competence for uptake of both 4OW-14 DNA and homologous DNA. An additional characteristic of homologous
DNA uptake in the B. subtilis system is the conversion of double-stranded DNA to single strands during transport into the cell (16). In the case of 4OW-14, analyses based on fractionation of cell lysates in CsCl gradients demonstrated that after uptake 4W-14 DNA molecules are in the single-stranded form and gradually disappear during incubation of the cells after uptake. This latter process is accompanied by association of the ,W-14 DNA label with recipient DNA, most likely as a result of incorporation of monomers, as occurs with other heterologous DNAs in B. subtilis (17) and Streptococcus pneumoniae (8). Consequently, notwithstanding the apparently additional different pathway of transport of 4W14 DNA, the behavior of this DNA after uptake, including its intracellular conversion, is similar to the behavior of homologous DNA. In this investigation we obtained information not only about the pathway of 4W-14 DNA
VOL. 149, 1982
uptake, but also about the affinity of 4W-14 DNA for homologous DNA receptors, which is lower than the affinity of homologous DNA (see above) and much higher than the affinities of glucosylated DNAs of phages T4 (14) and T6 (18). Relevant to the foregoing is the observation that affinity for homologous DNA receptors is virtually unaltered after acetylation of QW-14 DNA (Table 2). It follows that both the charge and the bulk of the putrescinyl side chain, as discussed previously (11), are probably of minor importance as regards the affinity for homologous DNA receptors; furthermore, the difference in behavior between phage T6 DNA and phage 4W-14 DNA must be ascribed to the difference in spatial configuration of the pyrimdine 5-substituents located in the major grooves of the respective DNA helices. In addition, the numbers of pyrimidine residues with 5-substituents in phage T6 DNA (where 13% of the residues are glucosylated) and in phage 4W-14 DNA (where 12.3% of the residues contain the putrescine moiety) are virtually the same. Hence, the steric hindrance due to bulky glucosyl residues is obviously greater than that due to the aliphatic putrescinyl chains. ACKNOWLEDGMENTS This research was supported by the Exchange Program of the Polish Academy of Sciences and the Consejo Superior de Investigaciones Cientificas, by grant 09.7.1 from Polish Academy of Sciences, and by Operating Grant A 3686 from the Natural Sciences and Engineering Research Council of Canada. LITERATURE CITED 1. Barnhart, B. J., and R. M. Herriott. 1963. Penetration of deoxyribonucleic acid into Hemophilus influenzae. Biochim. Biophys. Acta 76:25-39. 2. Cahn, F. H., and M. S. Fox. 1968. Fractionation of transformable bacteria from competent cultures of Bacillus subtilis on Renografin gradients. J. Bacteriol. 95:867878. 3. Cato, A., 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. Mal. Biol. 37:157-178. 4. Dougherty, T. J., A. Asmus, and A. Tomasz. 1979. Specificity of DNA uptake in genetic transformation of gonococci. Biochem. Biophys. Res. Commun. 86:97-107.
UPTAKE AND FATE OF fW-14 DNA
605
5. Garcia, E., P. Lopez, M. T. Perez Urena, and M. Espinosa. 1978. Early stages in Bacillus subtilis transformation: association between homologous deoxyribonucleic acid and surface structures. J. Bacteriol. 135:731-740. 6. KeUn, R. A., and R. A. J. Warren. 1973. Studies on the biosynthesis of a-putrescinylthymine in bacteriophage 4W-14-infected Pseudomonas acidovorans. J. Virol. 12:1427-1433. 7. Kropinskli, A. M. B., R. J. Bose, and R. A. J. Warren. 1973. 544-Aminobutylaminomethyl)uracil, and unusual pyrimidine from the deoxyribonucleic acid of bacteriophage 4W-14. Biochemistry 12:151-157. 8. Lacks, S., B. Greenberg, and K. Carlson. 1967. Fate of donor DNA in pneumococcal transformation. J. Mol. Biol. 29:327-347. 9. Lacks, S. A. 1977. Binding and entry of DNA in bacterial transformation, p. 179-219. In J. L. Reissig (ed.), Microbial interactions, vol. 3. Chapman and Hall, London. 10. Lewis, H. A., R. C. Miller, Jr., J. C. Stone, and R. A. J. Warren. 1975. Alkali lability of bacteriophage OW-14 DNA. J. Virol. 16:1375-1379. 11. Lopez, P., M. Espinosa, M. Piechowska, and D. Shugar. 1980. Influence of bacteriophage PBS1 and 4W-14 deoxyribonucleic acids on homologous deoxyribonucleic acid uptake and transformation in competent Bacillus subtilis. J. Bacteriol. 143:50-58. 12. Lopez, P., M. Espinosa, M. Plechowska, D. Shugar, and R. A. J. Warren. 1981. 4W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA. J. Virol. 37:559-563. 13. Lopez, P., M. Espinosa, R. A. J. Warren, M. Plechowska, and D. Shugar. 1981. Competition by phage 4)W-14 DNA of transforming DNA uptake, and its influence on transformation and transfection, in Bacillus subtilis. In Transformation 1980. Oxford, Cotswold. 14. Lopez, P., M. T. Perez Urena, E. Garcia, and M. Espinosa. 1980. Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis. J. Bacteriol. 142:229-235. 15. Notani, N. K., and J. K. Setlow. 1974. Mechanism of bacterial transformation and transfection. Prog. Nucleic Acid Res. Mol. Biol. 14:39-100. 16. Piechowska, M., and M. S. Fox. 1971. Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bacteriol. 108:680-689. 17. Plechowska, M., A. Soltyk, and D. Shupr. 1975. Fate of heterologous deoxyribonucleic acid in Bacillus subtilis. J. Bacteriol. 122:610-622. 18. Soltyk, A., D. Shugar, and M. Peichowska. 1975. Heterologous deoxyribonucleic acid uptake and complexing with cellular constituents in competent Bacillus subtilis. J. Bacteriol. 124:1429-1438. 19. Splizen, J., B. E. Reilly, and A. H. Evans. 1966. Microbial transformation and transfection. Annu. Rev. Microbiol. 20:371-400. 20. Young, F. E., and J. Splzizen. 1963. Incorporation of deoxyribonucleic acid in the Bacillus subtilis transformation system. J. Bacteriol. 86:392-400.
