Vol. 122, No. 3
JouRNAL or BACTErIOLoGY, June 1975, p. 987-993 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Transformation of Bacillus subtilis: Transforming Ability of Deoxyribonucleic Acid in Lysates of L-Forms or Protoplasts GEORGE E. BETTINGER AND FRANK E. YOUNG* Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 25 November 1974
The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.
The bulk of the studies on deoxyribonucleic acid (DNA)-mediated transformation of Bacillus subtilis utilize DNA which has been extracted from cells, partially purified, and fragmented into segments varying in length up to 6 x 107 daltons (19). The method of DNA isolation is known to affect the ability to demonstrate linkage between markers on donor DNA (18), as well as the ability to recover highmolecular-weight single-stranded DNA of donor origin in transformed cells (6). The direct use of DNA released by protoplast lysis for transformation has been mentioned (18, 19), but in only one instance was the efficiency of this donor DNA compared to DNA isolated by conventional techniques. Under these conditions the DNA remaining bound to membrane fragments after disruption was found to be two to three times more efficient (23). When the DNA made available by gently lysing a stable L-form of B. subtilis (28) was used to transform competent cells, we found that the frequency of transformation for single markers was approximately 10-fold greater than when DNA was isolated by phenol extraction (2). We postulated that the nature of the DNA as it exists in the L-form lysate before extraction was responsible for its greater efficiency. In addition, the DNA in L-form lysates was also able to co-transform two, three, and even four widely separated markers at frequencies far in excess of those predicted by congression. When B. subtilis protoplasts are carefully lysed, the entire chro-
mosome remains intact (17) and attached to a membrane fragment. In view of the endwise attachment (10) and linear uptake of transforming DNA (25, 26), we suggested that the high frequency of multiple marker transformations resulted from the ordered entry of markers on one strand of DNA liberated by gently lysing the L-form population. In the results reported here, we have tested these hypotheses using L-form lysates and extended our observations to DNA in lysates prepared from protoplasts of B. subtilis. MATERIALS AND METHODS Bacterial strains and culture conditions. The strains of B. subtilis used in this study, and their origin, are listed in Table 1. Bacillary strains were grown 24 h at 37 C on tryptose blood agar base (Difco, Detroit, Mich.) and then stored at room temperature. Stocks were periodically transferred and held at least 1 week prior to use as inocula for the development of competence. A stable L-form (MA) of B. subtilis BR151 isolated by Young et al. (28) was maintained in an L-form medium (LM) containing (per liter of salts solution described by Spizizen [24]): casein hydrolysate (Difco), 200 mg; MgSO4 .7H20, 5 mg; L-tryptophan, L-lysine, and L-methionine, 50 mg each; NaCl, 70 g; and glucose (autoclaved separately), 5 g. Transformation. Auxotrophic strains of B. subtilis were grown to competence using the modifications of the method of Erickson and Copeland (12) as described by Boylan et al. (3), and transformed immediately or otherwise stored at -70 C in 15% (wt/vol) of glycerol before transformation. Two sources of transforming DNA were used: (i) phenol-extracted DNA,
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BETTINGER AND YOUNG
prepared as outlined by Yasbin and Young (27) and termed isolated DNA; or (ii) DNA made available by the gentle lysis of an L-form or protoplast suspension and referred to as lysate DNA. Competent cells were exposed to DNA for 30 min at 37 C, washed twice with Spizizen salts (24), and resuspended to between 0.5 to 0.1 their original volume. Dilutions were plated for both transformants and viable count on minimal agar plus auxotrophic supplements (3). Preparation of lysate DNA. L-form lysate DNA was prepared by adding 1 volume of an L-form suspension to a screw-capped tube (13 by 100 mm) and then slowly adding an equal volume of Spizizen salts. Three volumes of competent cells were added, and the tube was slowly inverted to mix the contents and then incubated at 50 rpm at 37 C. Protoplast lysate DNA was similarly obtained by aseptically preparing protoplasts as outlined by Sargent et al. (22) from an overnight culture of B. subtilis grown in antibiotic medium 3 (Difco). Protoplasts were sedimented at 4,000 x g for 10 min at room temperature, washed once with the LM medium, and resuspended in that medium to an optical density at 585 nm (OD.85) of 0.400. When isolated DNA was used for transformation it was first diluted in minimal salts to a predetermined DNA concentration. This was then mixed with an equal volume of LM medium, and finally 3 volumes of competent cells were added. In this way the salt concentrations in the final transformation mixture were comparable to transformations with lysate DNA, and the DNA concentration could be regulated. DNA measurements. Isolated DNA was estimated by the method of Burton (4) using calf thymus DNA as a standard. The cellular DNA content of the L-form was determined by adding 50 uCi of [methyl8H ]thymidine (18 Ci/mMol) to 50 ml of an overnight L-form culture (OD58, = 0.420) and incubating at 37 C for 2 h (OD,8. = 0.470). Nonlabeled thymidine was then added at a 100-fold excess, and 5 ml of the culture transferred to an equal volume of cold 10% trichloroacetic acid. The precipitate formed after 3 h was collected onto a glass-fiber filter, washed with cold 5% trichloroacetic acid, and counted when dry. One-half milliliter of the remaining 45-ml culture was diluted 10-fold in 8.5% (wt/vol) NaCl containing 1.0% (wt/vol) NaN,, and the number of L-form particles was estimated by direct counting using a PetroffHauser chamber (5). The remaining 44.5 ml of culture was sedimented and resuspended in 5 ml of Spizizen minimal salts, and the DNA was isolated by phenol extraction (27). The specific activity of the radioactive phenol extracted L-form DNA was determined by precipitating 20 ug of this DNA in the presence of 100 ;g of unlabeled calf thymus carrier DNA on fiber filters and by determining the radioactivity. The MA L-form grown in LM medium contains approximately 1.5 x 10-8 ;g of DNA per L-form particle, based on isolating DNA of a specific activity of 22.3 counts/min per ;g of DNA from a [3H]thymidine-labeled culture having 2.85 x 108 particles/ml and 116 3H counts/min per ml of cold 5% trichloroacetic acid-precipitable material. This estimate was subsequently used in adjusting the level of transforming DNA in L-form lysates.
J. BACTERIOL.
Measurement of radioactivity. Radioactive samples were counted in 5 ml of a commercial toluenebased scintillation cocktail (Omnifluor) in a Beckman LS-230 liquid scintillation counter. Materials. [Methyl-3H]thymidine and Omnifluor were obtained from New England Nuclear Corp., Boston, Mass. Glass-fiber filters (934 AH) were purchased from Reeve Angel, Clifton, N.J. Deoxyribonuclease (DNase) and ribonuclease (RNase) were products of Worthington Biochemicals, Freehold, N.J. Pronase, trypsin, and Aquacide II were purchased from Calbiochem, La Jolla, Calif.
RESULTS Transformation by L-form lysate DNA. Transformation of RUB125 by L-form lysate DNA was 10 times more efficient in transforming single markers than isolated DNA, even though the concentration of isolated DNA was three times that of the lysate DNA. The occurrence of multiple marker transformations using L-form lysate DNA is greater than when the corresponding isolated DNA is used (2). Comparable results are observed when strains SB202 and RUB783 are transformed in the same manner (unpublished data). Since the isolation of DNA reduced both the transformation efficiency and the ability to transform multiple markers, the L-form lysate was pretreated for 30 min with either RNase, pronase (both used in the DNA extraction procedure), trypsin, or DNase prior to the addition of competent cells to see whether the transforming properties of the lysate DNA could be selectively diminished. The data (Table 2) show that only DNase markedly affected the transforming ability of L-form lysate DNA, reducing single marker transformation by three logarithms, whereas multiple marker transformations were even more sensitive. We next examined the effect of the lysate on Met+ transforming ability of isolated DNA to determine whether the lysate in some way rendered DNA more suitable for transformation. Two concentrations of isolated RUB818 DNA were mixed with the L-form lysate, with the ratio of the isolated RUB818 DNA to L-form lysate DNA expressed as RDNA. In these experiments Met+ could only originate from the isolated DNA. When RDNA was 2.0 the Met+ transformation of BR151 was 0.52%, but it decreased to 0.10% if both recipient cells and isolated DNA were simultaneously added to a lysate DNA preparation. Pretreatment of the isolated DNA in the L-form lysate for 15 min prior to the addition of competent cells resulted in an increase in Met+ transformants from 0.10 to 0.17%. However, in both instances in which lysate DNA was present, Met+ transformation
Vol.. 122, 1975
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TRANSFORMATION BY CRUDE LYSATE DNA TABLE 1. Strains of B. subtilis Genotype or phenotype
Source
BR57 BR99 BR151 SB202 RUB321 RUB783 RUB818 RUB122 RUB125
thr-2, ura-1, trpC2 ura-1, trpC2, ser-1 Iys-3, trpC2, metBlO aroB2, trpC2, hisB2, tyrAl Iys-3, trpC2, hisB2 RfmR, purB6, hisAl, leuA8, metBlO Prototroph of BR151 RfmR, purB6, hisAl, ura-1, leuA8 thr-2, hisAl, ura-1, leuA8
RUB128 RUB129 RUB130 RUB131 RUB132 MA L-form
lys-3, trpC2, hisB2, RfmR hisAl, ura-1, IeuA8 thr-2, ura-1, leuA8 thr-2, hisAl, IeuA8 thr-2, hisAl, ura-1 metBlO
B. E. Reilly B. E. Reilly B. E. Reilly A. Ganesan G. A. Wilson U. Streips R. E. Yasbin (RUB783) x [BR991G (RUB122) x [BR57 la RfmR spontaneously lost Spontaneous from RUB321 (RUB125) x IRUB8181]b (RUB125) x [RUB818]b (RUB125) x [RUB818]b (RUB125) x [RUB818]" Subculture of sal-lc
Designation
a By congression, notation = (recipient cell) x [donor DNA]. b By transformation, notation = (recipient cell) x [donor DNA]. c Originally subcultured from sal-1 isolated by Young et al. from BR151 (28) in LM medium plus lysine, tryptophan, and methionine, but now Lys+Trp+ as evidenced by its ability to transform BR151 to Lys+ and Trp+ but not Met+.
TABLE 2. Effect of nuclease and protease oz L-form lysate DNAa Selected marker(s) Treatment
Control DNase (50 ug/ml) RNase (250 Ag/ml) Pronase (500,gg/ml) Trypsin (500 jg/ml)
Enzyme/DNA ratio
0 9.4 47.0 94.0 94.0
Thr+
Thr+His+
Thr+His+Ura+
1.000b 0.001 0.725 0.945 0.930
1.00Oa 0.0001 0.755 1.150 0.875
1.000" 0.000001 0.920 1.500 1.090
a L-form lysate DNA was prepared as outlined in Materials and Methods and contained 5.3 ,ug of DNA/ml. The lysates were enzymatically treated for 30 min at 37 C prior to the addition of the RUB125 recipient. "Results expressed as the number of transformants relative to the control value, reported as unity.
was 70 to 80% less than when isolated DNA was used alone, and still approximately 10-fold less than expected for lysate DNA (Trp+ transformation by lysate DNA was 1.74% in this experiment, data not shown). When the RDNA was 6.2, Met+ transformation by isolated DNA alone was 2.0%, but again decreased approximately 50% if lysate DNA was present. Pretreatment of the isolated DNA either by an L-form lysate or pronase-digested L-form lysate did not significantly affect the transforming ability of the isolated DNA (Table 3). Transformation by protoplast lysate DNA. The data in Tables 2 and 3 suggested that there was nothing peculiar in the MA L-form DNA preparation or in the lysate that resulted in the increased transformation efficiency and frequencies of multiple marker transformation observed with lysate DNA. Transformation by isolated L-form DNA was indistinguishable from DNA isolated from bacillary strains and
the transformation efficiency of isolated DNA was not stimulated by the L-form lysate (Table 3). If the physical state of the lysate DNA were responsible for the increased transformation, results similar to those found using the L-form lysate DNA might also be obtained by transformation using a lysate prepared from protoplasts of bacillary cells. Gently lysed protoplasts of B. subtilis, prepared as outlined above, were relatively comparable to L-form lysate DNA in their ability to multiply transform RUB125, as shown in Table 4, although the efficiency of single marker transformation by protoplast lysate DNA was generally one-half to threefourths that obtained with an L-form lysate (data not shown). Unselected marker transformation. The linkage between trpC2 and hisB2 by transformation is normally 50% with isolated DNA (20). Transformation of these markers by the L-form lysate DNA (RUB321 recipient) and by the
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J. BACRIOL.
BETTINGER AND YOUNG TABLE 3. Effect of MA L-form lysate on transformation of BR151 by Isolated DNAa Transformation components added at:"
Met+ transformants
Expt
0 min
1
L-form lysate, isolated DNAC L-form lysate
L-form lysate 2
15 min
30 min
45 min
(%)
BR151 cells
0.17
BR151 cells isolated DNAC BR151 cells, isolated DNAc BR151 cells
0.10
0.52 0.0
L-form lysate, pronase L-form lysate
isolated DNAd
BR151 cells
1.5 1.0
L-form lysate L-form lysate
isolated DNAd
BR151 cells, isolated DNAd BR151 cells BR151 cells, isolated DNAd BR151 cells
0.9 1.1
BR151 cells BR151 cells BR151 cells
2.0 0.0 0.0
L-form lysate, pro-
0.0
nase
isolated DNAd
Pronase L-form lysate
a Competent BR151 (lys-3,trpC2,metB1O) cells and isolated RUB818 DNA (prototrophic) were prepared as described in the text, and transformation was performed as outlined therein. Pronase was used at 0.5 mg/ml final concentration after addition to L-form lysate or isolated DNA in the transformation tube. The DNA in experiment 2 was prepared similarly to experiment 1 except it was concentrated by Aquacide II in dialysis tubing. b The L-form lysate and isolated DNA were added to the transformation tube at the times indicated to effect a pretreatment prior to the addition of competent cells, after which transformation was allowed to proceed for
30 min. c L-form DNA present at 1 Ag/ml, isolated DNA at 2 jig/ml (fmial concentrations after BR151 competent cell
addition). (RUB818 DNAIL-form DNA, 2.) dL-form DNA present at 0.8 sg/ml, isolated DNA at 5 jg/ml (final concentrations after BR151 competent cell addition). (RUB818 DNA/L-form DNA, 6.2.) TABLE 4. Transformability of RUB125 by gently Iysed L-forms or BR151 protoplastsa Relative transformation of RUB125 by donor lysate DNA preparations
Selected marker(s)
Thr His Ura Leu Thr His His Ura Ura Leu Thr His Ura His Ura Leu Thr His Ura Leu
L-form lysate DNA
BRi51 proto-
1.00 1.10 0.83 0.68 0.11 0.08 0.14 0.01 0.01 0.002
1.00 0.63 0.78 0.71 0.10 0.09 0.08 0.02 0.01 0.003
plast lysate DNA
aA culture of competent RUB125 (thr-2, hisAl,
ura-1, leuA8) was transformed either by gently lysed MA L-forms or BR151 protoplasts as outlined in the Methods. The results are shown relative to Thr+ transformants.
protoplast lysate DNA (RUB128 recipient) showed co-transformation of the trpC2 hisB2 markers at higher frequencies, approaching 100% (1; Table 5). This region is expanded in the SB202 strain (aroB2, trpC2, hisB2, tyrAl). When SB202 was transformed using L-form lysate DNA and Aro+ transformants were selected, the other markers were co-transformed in the order: Trp+ (82%), His+ (77%), and Tyr+ (72%) with Aro+ normalized to 100%. These data are relatively similar if isolated DNA is used, only the co-transformation frequencies were lower as predicted. When Tyr+ was selected in a transformation by lysate DNA, the nonselected, co-transformation of any individual marker or all three was greater than 91%. Dilution analysis of multiple marker transformation. Although the frequencies of multiple marker transformations listed in Table 5 (and reference 2) exceed those expected for congression (13), this does not prove that these genes are taken in on the same fragment of transforming DNA. As discussed by Goodgal
TRANSFORMATION BY CRUDE LYSATE DNA
VOL. 122, 1975
TABLE 5. Co-transformation of non-selected markersa
Recipient
Donor
% Nonselected Select- Nonselect- marker trscoed ed maformaker f°tion
makr
for:
L-form lysate DNA RUB128 RUB818 protoplast lysate DNA
Trp His
89
His Trp
94
SB202
Aro Trp
82
RUB321
L-form lysate DNA
HisB TyrA Trp His
77 72 69
Tyr
SB202
L-form isolated DNA
Aro Trp
His Tyr Trp His
65 53 52 48
991
loci are known to exist on the same chromosome; and a mixture of strains RUB129 (hisAl, ura-1, leuA8), RUB130 (thr-2, ura-1, leuA8), and RUB131 (thr-2, hisAl, leuA8) protoplasts, in which the prototrophic traits under investigation are on different chromosomes. The latter three strains (RUB129, RUB130, and RUB131) were protoplasted separately, adjusted to the same OD,,, of the BR151 protoplasts, and mixed in equal volumes. Serial dilutions of the BR151 protoplasts as well as the protoplast mixture were made, the diluted protoplasts were lysed, and competent RUB132 cells were added. Nonprotoplasted cells were selected against by elimination of leucine and methionine from the minimal medium. The results (Fig. 1) show that BR151 protoplasts (Thr+Ura+His+) transform RUB132 for one, two, or three markers at constant ratios, independent of DNA dilution, but that the frequency of multiple marker transformants ob106
Tyr
SB202
L-form lysate DNA
Tyr Aro
98
Trp His
91 95 91
AroTrp His
aTransformation of competent cells was as described in Materials and Methods. Nonselected transformation markers were supplemented for in the initial transformation isolation and detected by subsequent streaking of isolated colonies onto differential media. The Tyr+ transformants were selected on plates supplemented with phenylalanine, tryptophan, and histidine. Shikimic acid was omitted since phenylalanine alone satisfies the aroB2 requirement. The results are expressed as the percent of 200 selected transformants showing the nonselected phenotype.
(14), if multiple marker transformants arise by the integration of the genes from different fragments of DNA, the frequency of multiple transformations will drop off more rapidly than the number of single transformants as a function of DNA dilution. Similarly, if these genes originate on the same strand, multiple transformants should constitute a constant fraction of single marker transformants upon dilution of DNA. To examine this question, strain RUB132 (carrying thr-2, hisAl, ura-1) was transformed for Thr+, Thr+His+, and Thr+His+Ura+ by protoplast lysate DNA. Two protoplast lysate DNA preparations were used: BR151 protoplasts (Iys-3, trpC2, metBlO), in which the
105 z 4U
LOG2 DILUTION
FIG. 1. Dilution analysis of multiple marker transformation. Serial dilutions of protoplast lysate DNA were used to transform cells of RUB132 (thr-2, hisAl, ura-1) with two preparations of DNA as described in the text. Donor DNA from protoplasts of strain BR151 (prototrophic for all selected traits): 0, Thr+; A, Thr+His+; and U, Thr+His+Ura+ transformants. Donor DNA from a mixture of protoplasts of strains RUB129, RUB130, and RUB131 (each selected trait from a different strain): 0, Thr+, A, Thr+His+; and 0, Thr+His+Ura+ transformants.
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J. BACTERIOL.
tained using the protoplast mixture drops off cell, since when present at only one-sixth the more rapidly with DNA dilution than does the concentration of isolated DNA it inhibits transfrequency for single marker transformation. formation by that DNA by 50%. It is known that after the gentle lysis of B. DISCUSSION subtilis protoplasts under conditions similar to B. subtilis 168 derivatives are transformed by ours, the chromosome remains intact and atDNA made available by the gentle lysis of tached to the cell membrane (17). We have either a stable L-form (MA) or protoplasts at postulated that the high co-transformation obfrequencies up to 10-fold greater than those served using DNA in the L-form lysate (and obtained with DNA isolated by the more con- protoplasts as well) was due to the uptake of the ventional procedures (2; Tables 4 and 5). The markers from a continuous donor DNA molemethod of DNA isolation used involves removal cule (2). The data in Fig. 1 conclusively prove of the cell wall by lysozyme, osmotic lysis of the our hypothesis. Thr+His+ and Thr+His+Ura+ protoplast, pronase digestion, solubilization of co-transformations of RUB132 (thr-2, hisAl, the cytoplasmic membrane by detergent, and ura-1) by BR151 protoplast lysate DNA (protofinally extraction with phenol. The remaining trophic for all markers transformed) is a conDNA is essentially free of protein and RNA. stant fraction of Thr+ transformants at all Hirokawa (16) reported that the level of trans- dilutions of DNA. These results are predicted if fection of B. subtilis by DNA isolated from all markers are taken up from the same, continbacteriophage 029 could be diminished by treat- uous segment of donor DNA (14). Conversely, ment with pronase, although pronase has no co-transformation of RUB132 for the same such effect on transfection with DNA from marker sets by DNA prepared by gently lysing a bacteriophage 44 (21). In view of this, and since mixture of protoplasts of strains RUB129, the lysate DNA preparations used for transfor- RUB130, and RUB131 (each prototrophic gene mation are essentially untreated, the possibility originates from a separate strain) behaves as arose that the loss of certain components during predicted by congression in that only Thr+ DNA isolation was responsible in some way for transformation is a linear function of dilution, the decreased transformation efficiency seen whereas Thr+His+ and Thr+His+Ura+ transforwith isolated DNA. The L-form lysate DNA, mants are not a constant fraction of the Thr+ however, proved refractory to attempts to di- transformants. When SB202 (aroB2, trpC2, minish its transformation efficiency by either hisBl, tyrAl) was transformed by lysate DNA RNase, pronase, or trypsin (Table 2). Even after and Aro+ transformants were selected, the coextensive DNase treatment the lysate retained transformation frequencies of the other markers significant transforming ability for single (0.1% decreased in the order of their replication. If of control) and double (0.01% of control) mark- these markers are integrated in the order of ers. As noted in the text, DNase had a more their replication as suggested by Erickson and pronounced effect on unlinking markers. The Braun (11), then by selecting SB202 transformsuperior biologic activity of the lysate DNA may ants for the marker last in the replication order reflect a more native physical nature. Snyder the frequency of co-transfer of the unselected and Young (23) reported that DNA still at- preceding markers should be similar and (as tached to membrane fragments prepared from shown in Table 5) they are all greater than 90% lysed protoplasts after rigorous homogenization co-transformed. Although this is a closely transformed at efficiencies two- to three-fold linked region even when transformed by isohigher than if the DNA were isolated by conven- lated DNA (Table 5), there is some indication of tional methods. The membrane fragments to polarity in the entrance and/or integration of which the DNA was attached might afford a these transforming genes. protective structural support. It also seems Transforming DNA is thought to attach to a reasonable to suggest that it is membrane competent cell in an endwise fashion (10) and attached DNA, or a fraction thereof, which in subsequently converted to membrane-bound, lysate preparations is resistant to DNase diges- double-strand fragments of approximately 9 x tion and retains the ability after exposure to 106 daltons by endonuclease (1, 7, 9, 15). AlDNase to transform competent cells. The L- though bound, the double-strand fragment is form lysate was tested for its ability to enhance further broken down into single-strand fragthe transforming efficiency of isolated DNA ments of 2.8 x 106 daltons, which complex with (Table 3). Overall, the data indicate that the the recipient genome and are integrated (8). lysate did not increase the transformation by Assuming that the lysate DNA is attached to isolated DNA. Lysate DNA does, however, seem the competent cell, taken up in a linear fashion, able to bind more effectively to the competent and fragmented as just described for isolated
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TRANSFORMATION BY CRUDE LYSATE DNA
DNA, the high incidence of multiple marker transformations observed using lysate DNA is then perhaps due to an ordered arrangement of membrane-bound, single-strand fragments, following linear uptake of a continuous DNA strand and a polarity of gene integration during chromosome replication (12, 13). ACKNOWLEDGMENTS We thank G. A. Wilson for his helpful discussions during the course of this work. This research was aided by grants from the American Cancer Society (VC 27-J) and from the United States Public Health Service (AI-10141) from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Arwert, F., and G. Venema. 1973. Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Mole. Gen. Genet. 123:185-198. 2. Bettinger, G. E., and F. E. Young. 1973. Transformation of Bacillus subtilis using gently lysed L-forms, a new mapping technique. Biochem. Biophys. Res. Commun.
55:1106-1111. 3. Boylan, R. J., N. H. Mendelson, D. Brooks, and F. E. Young. 1972. Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J. Bacteriol. 110:281-290. 4. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J.
62:315-323. 5. Chatterjee, A. N., H. Taber, and F. E. Young. 1971. A rapid method for synchronization of Staphylococcus aureus and Bacillus subtilis. Biochem. Biophys. Res. Commun. 44:1125-1130. 6. 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.
116:146-153. 7. Dooley, D. C., and E. W. Nester. 1973. Deoxyribonucleic acid-membrane complexes in the Bacillus subtilis transformation system. J. Bacteriol. 114:711-722. 8. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: size and distribution of the integrated donor segments. J. Bacteriol. 111:488-494. 9. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming DNA following uptake by competent Bacillus subtilis. Ill. Formation and properties of products isolated from transformed cells which are derived entirely from donor DNA. J. Mol. Biol. 64:9-29. 10. Dubnau, D., and C. Cirigliano. 1972. Fate of transforming DNA following uptake by competent Bacillus subtilis. IV. The endwise attachment and uptake of transforming DNA. J. Mol. Biol. 64:31-46. 11. Erickson, R. J., and W. Braun. 1968. Apparent dependence of transformation on the state of deoxyribonucleic acid replication of recipient cells. Bacteriol. Rev.
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32:291-296. 12. Erickson, R. J., and J. C. Copeland. 1972. The structure and replication of chromosomes in competent cells of Bacillus subtilis. J. Bacteriol. 109:1075-1084. 13. Erickson, R. J., and J. C. Copeland. 1973. Congression of unlinked markers and genetic mapping in the transformation of Bacillus subtilis 168. Genetics 73:13-21. 14. Goodgal, S. 1961. Studies on transformations of Hemophilus influenzae. IV. Linked and unlinked transformation. J. Gen. Physiol. 45:205-228. 15. Harris, W. J., and G. C. Barr. 1971. Mechanism of transformation in Bacillus subtilis. Mole. Gen. Genet. 113:331-344. 16. Hirokawa, H. 1972. Transfecting deoxyribonucleic acid of Bacillus bacteriophage 029 that is protease sensitive. Proc. Natl. Acad. Sci. U.S.A. 69:1555-1559. 17. Ivarie, R. D., and J. J. Pene. 1970. Association of the Bacillus subtilis chromosome with the cell membrane: resolution of free and bound deoxyribonucleic acid on Renografin gradients. J. Bacteriol. 104:839-850. 18. Kelly, M. S., and R. H. Pritchard. 1965. Unstable linkage between genetic markers in transformation. J. Bacteriol. 89:1314-1321. 19. Levin, B. C., and 0. E. Landman. 1973. DNA synthesis inhibition by 6-(p-hydroxyphenylazo)-uracil in relation to uptake and integration of transforming DNA in Bacillus subtilis, p. 217-240. In L J. Archer (ed.), Bacterial transformation. Academic Press Inc., New York. 20. Nester, E., and J. Lederberg. 1961. Linkage of genetic units of Bacillus subtilis in DNA transformation. Proc. Natl. Acad. Sci. U.S.A. 47:52-55. 21. Reilly, B. E., and J. Spizizen. 1965. Bacteriophage deoxyribonucleate infection of competent Bacillus subtilis. J. Bacteriol. 89:782-790. 22. Sargent, M. G., B. K. Ghosh, and J. 0. Lampen. 1968. Characteristics of penicillinase release by washed cells of Bacillus licheniformis. J. Bacteriol. 96:1231-1239. 23. Snyder, R W., and F. E. Young. 1969. Association between the chromosome and the cytoplasmic membrane in Bacillus subtilis. Biochem. Biophys. Res. Commun. 35:354-362. 24. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S.A. 44:1072-1078. 25. Strauss, N. 1965. Configuration of transforming deoxyribonucleic acid during entry into Bacillus subtilis. J. Bacteriol. 89:288-293. 26. Strauss, N. 1966. Further evidence concerning the configuration of transforming deoxyribonucleic acid during entry into Bacillus subtilis. J. Bacteriol. 91:702-708. 27. Yasbin, R. E., and F. E. Young. 1972. The influence of temperate bacteriophage 0105 on transformation and transfection in Bacillus subtilis. Biochem. Biophys. Res. Commun. 47:365-371. 28. Young, F. E., P. Haywood, and M. Pollock. 1970. Isolation of L-forms of Bacillus subtilis which grow in liquid medium. J. Bacteriol. 102:867-870. 29. Young, F. E., and G. A. Wilson. 1972. Genetics of Bacillus subtilis and other gram positive sporulating bacilli, p. 77-106. In H. 0. Halvorson, R. Hanson, and L. L. Campbell (ed,), Spores V. American Society for Microbiology, Washington, D. C.
JouRNAL or BACTErIOLoGY, June 1975, p. 987-993 Copyright 0 1975 American Society for Microbiology
Printed in U.S.A.
Transformation of Bacillus subtilis: Transforming Ability of Deoxyribonucleic Acid in Lysates of L-Forms or Protoplasts GEORGE E. BETTINGER AND FRANK E. YOUNG* Department of Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 Received for publication 25 November 1974
The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.
The bulk of the studies on deoxyribonucleic acid (DNA)-mediated transformation of Bacillus subtilis utilize DNA which has been extracted from cells, partially purified, and fragmented into segments varying in length up to 6 x 107 daltons (19). The method of DNA isolation is known to affect the ability to demonstrate linkage between markers on donor DNA (18), as well as the ability to recover highmolecular-weight single-stranded DNA of donor origin in transformed cells (6). The direct use of DNA released by protoplast lysis for transformation has been mentioned (18, 19), but in only one instance was the efficiency of this donor DNA compared to DNA isolated by conventional techniques. Under these conditions the DNA remaining bound to membrane fragments after disruption was found to be two to three times more efficient (23). When the DNA made available by gently lysing a stable L-form of B. subtilis (28) was used to transform competent cells, we found that the frequency of transformation for single markers was approximately 10-fold greater than when DNA was isolated by phenol extraction (2). We postulated that the nature of the DNA as it exists in the L-form lysate before extraction was responsible for its greater efficiency. In addition, the DNA in L-form lysates was also able to co-transform two, three, and even four widely separated markers at frequencies far in excess of those predicted by congression. When B. subtilis protoplasts are carefully lysed, the entire chro-
mosome remains intact (17) and attached to a membrane fragment. In view of the endwise attachment (10) and linear uptake of transforming DNA (25, 26), we suggested that the high frequency of multiple marker transformations resulted from the ordered entry of markers on one strand of DNA liberated by gently lysing the L-form population. In the results reported here, we have tested these hypotheses using L-form lysates and extended our observations to DNA in lysates prepared from protoplasts of B. subtilis. MATERIALS AND METHODS Bacterial strains and culture conditions. The strains of B. subtilis used in this study, and their origin, are listed in Table 1. Bacillary strains were grown 24 h at 37 C on tryptose blood agar base (Difco, Detroit, Mich.) and then stored at room temperature. Stocks were periodically transferred and held at least 1 week prior to use as inocula for the development of competence. A stable L-form (MA) of B. subtilis BR151 isolated by Young et al. (28) was maintained in an L-form medium (LM) containing (per liter of salts solution described by Spizizen [24]): casein hydrolysate (Difco), 200 mg; MgSO4 .7H20, 5 mg; L-tryptophan, L-lysine, and L-methionine, 50 mg each; NaCl, 70 g; and glucose (autoclaved separately), 5 g. Transformation. Auxotrophic strains of B. subtilis were grown to competence using the modifications of the method of Erickson and Copeland (12) as described by Boylan et al. (3), and transformed immediately or otherwise stored at -70 C in 15% (wt/vol) of glycerol before transformation. Two sources of transforming DNA were used: (i) phenol-extracted DNA,
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BETTINGER AND YOUNG
prepared as outlined by Yasbin and Young (27) and termed isolated DNA; or (ii) DNA made available by the gentle lysis of an L-form or protoplast suspension and referred to as lysate DNA. Competent cells were exposed to DNA for 30 min at 37 C, washed twice with Spizizen salts (24), and resuspended to between 0.5 to 0.1 their original volume. Dilutions were plated for both transformants and viable count on minimal agar plus auxotrophic supplements (3). Preparation of lysate DNA. L-form lysate DNA was prepared by adding 1 volume of an L-form suspension to a screw-capped tube (13 by 100 mm) and then slowly adding an equal volume of Spizizen salts. Three volumes of competent cells were added, and the tube was slowly inverted to mix the contents and then incubated at 50 rpm at 37 C. Protoplast lysate DNA was similarly obtained by aseptically preparing protoplasts as outlined by Sargent et al. (22) from an overnight culture of B. subtilis grown in antibiotic medium 3 (Difco). Protoplasts were sedimented at 4,000 x g for 10 min at room temperature, washed once with the LM medium, and resuspended in that medium to an optical density at 585 nm (OD.85) of 0.400. When isolated DNA was used for transformation it was first diluted in minimal salts to a predetermined DNA concentration. This was then mixed with an equal volume of LM medium, and finally 3 volumes of competent cells were added. In this way the salt concentrations in the final transformation mixture were comparable to transformations with lysate DNA, and the DNA concentration could be regulated. DNA measurements. Isolated DNA was estimated by the method of Burton (4) using calf thymus DNA as a standard. The cellular DNA content of the L-form was determined by adding 50 uCi of [methyl8H ]thymidine (18 Ci/mMol) to 50 ml of an overnight L-form culture (OD58, = 0.420) and incubating at 37 C for 2 h (OD,8. = 0.470). Nonlabeled thymidine was then added at a 100-fold excess, and 5 ml of the culture transferred to an equal volume of cold 10% trichloroacetic acid. The precipitate formed after 3 h was collected onto a glass-fiber filter, washed with cold 5% trichloroacetic acid, and counted when dry. One-half milliliter of the remaining 45-ml culture was diluted 10-fold in 8.5% (wt/vol) NaCl containing 1.0% (wt/vol) NaN,, and the number of L-form particles was estimated by direct counting using a PetroffHauser chamber (5). The remaining 44.5 ml of culture was sedimented and resuspended in 5 ml of Spizizen minimal salts, and the DNA was isolated by phenol extraction (27). The specific activity of the radioactive phenol extracted L-form DNA was determined by precipitating 20 ug of this DNA in the presence of 100 ;g of unlabeled calf thymus carrier DNA on fiber filters and by determining the radioactivity. The MA L-form grown in LM medium contains approximately 1.5 x 10-8 ;g of DNA per L-form particle, based on isolating DNA of a specific activity of 22.3 counts/min per ;g of DNA from a [3H]thymidine-labeled culture having 2.85 x 108 particles/ml and 116 3H counts/min per ml of cold 5% trichloroacetic acid-precipitable material. This estimate was subsequently used in adjusting the level of transforming DNA in L-form lysates.
J. BACTERIOL.
Measurement of radioactivity. Radioactive samples were counted in 5 ml of a commercial toluenebased scintillation cocktail (Omnifluor) in a Beckman LS-230 liquid scintillation counter. Materials. [Methyl-3H]thymidine and Omnifluor were obtained from New England Nuclear Corp., Boston, Mass. Glass-fiber filters (934 AH) were purchased from Reeve Angel, Clifton, N.J. Deoxyribonuclease (DNase) and ribonuclease (RNase) were products of Worthington Biochemicals, Freehold, N.J. Pronase, trypsin, and Aquacide II were purchased from Calbiochem, La Jolla, Calif.
RESULTS Transformation by L-form lysate DNA. Transformation of RUB125 by L-form lysate DNA was 10 times more efficient in transforming single markers than isolated DNA, even though the concentration of isolated DNA was three times that of the lysate DNA. The occurrence of multiple marker transformations using L-form lysate DNA is greater than when the corresponding isolated DNA is used (2). Comparable results are observed when strains SB202 and RUB783 are transformed in the same manner (unpublished data). Since the isolation of DNA reduced both the transformation efficiency and the ability to transform multiple markers, the L-form lysate was pretreated for 30 min with either RNase, pronase (both used in the DNA extraction procedure), trypsin, or DNase prior to the addition of competent cells to see whether the transforming properties of the lysate DNA could be selectively diminished. The data (Table 2) show that only DNase markedly affected the transforming ability of L-form lysate DNA, reducing single marker transformation by three logarithms, whereas multiple marker transformations were even more sensitive. We next examined the effect of the lysate on Met+ transforming ability of isolated DNA to determine whether the lysate in some way rendered DNA more suitable for transformation. Two concentrations of isolated RUB818 DNA were mixed with the L-form lysate, with the ratio of the isolated RUB818 DNA to L-form lysate DNA expressed as RDNA. In these experiments Met+ could only originate from the isolated DNA. When RDNA was 2.0 the Met+ transformation of BR151 was 0.52%, but it decreased to 0.10% if both recipient cells and isolated DNA were simultaneously added to a lysate DNA preparation. Pretreatment of the isolated DNA in the L-form lysate for 15 min prior to the addition of competent cells resulted in an increase in Met+ transformants from 0.10 to 0.17%. However, in both instances in which lysate DNA was present, Met+ transformation
Vol.. 122, 1975
989
TRANSFORMATION BY CRUDE LYSATE DNA TABLE 1. Strains of B. subtilis Genotype or phenotype
Source
BR57 BR99 BR151 SB202 RUB321 RUB783 RUB818 RUB122 RUB125
thr-2, ura-1, trpC2 ura-1, trpC2, ser-1 Iys-3, trpC2, metBlO aroB2, trpC2, hisB2, tyrAl Iys-3, trpC2, hisB2 RfmR, purB6, hisAl, leuA8, metBlO Prototroph of BR151 RfmR, purB6, hisAl, ura-1, leuA8 thr-2, hisAl, ura-1, leuA8
RUB128 RUB129 RUB130 RUB131 RUB132 MA L-form
lys-3, trpC2, hisB2, RfmR hisAl, ura-1, IeuA8 thr-2, ura-1, leuA8 thr-2, hisAl, IeuA8 thr-2, hisAl, ura-1 metBlO
B. E. Reilly B. E. Reilly B. E. Reilly A. Ganesan G. A. Wilson U. Streips R. E. Yasbin (RUB783) x [BR991G (RUB122) x [BR57 la RfmR spontaneously lost Spontaneous from RUB321 (RUB125) x IRUB8181]b (RUB125) x [RUB818]b (RUB125) x [RUB818]b (RUB125) x [RUB818]" Subculture of sal-lc
Designation
a By congression, notation = (recipient cell) x [donor DNA]. b By transformation, notation = (recipient cell) x [donor DNA]. c Originally subcultured from sal-1 isolated by Young et al. from BR151 (28) in LM medium plus lysine, tryptophan, and methionine, but now Lys+Trp+ as evidenced by its ability to transform BR151 to Lys+ and Trp+ but not Met+.
TABLE 2. Effect of nuclease and protease oz L-form lysate DNAa Selected marker(s) Treatment
Control DNase (50 ug/ml) RNase (250 Ag/ml) Pronase (500,gg/ml) Trypsin (500 jg/ml)
Enzyme/DNA ratio
0 9.4 47.0 94.0 94.0
Thr+
Thr+His+
Thr+His+Ura+
1.000b 0.001 0.725 0.945 0.930
1.00Oa 0.0001 0.755 1.150 0.875
1.000" 0.000001 0.920 1.500 1.090
a L-form lysate DNA was prepared as outlined in Materials and Methods and contained 5.3 ,ug of DNA/ml. The lysates were enzymatically treated for 30 min at 37 C prior to the addition of the RUB125 recipient. "Results expressed as the number of transformants relative to the control value, reported as unity.
was 70 to 80% less than when isolated DNA was used alone, and still approximately 10-fold less than expected for lysate DNA (Trp+ transformation by lysate DNA was 1.74% in this experiment, data not shown). When the RDNA was 6.2, Met+ transformation by isolated DNA alone was 2.0%, but again decreased approximately 50% if lysate DNA was present. Pretreatment of the isolated DNA either by an L-form lysate or pronase-digested L-form lysate did not significantly affect the transforming ability of the isolated DNA (Table 3). Transformation by protoplast lysate DNA. The data in Tables 2 and 3 suggested that there was nothing peculiar in the MA L-form DNA preparation or in the lysate that resulted in the increased transformation efficiency and frequencies of multiple marker transformation observed with lysate DNA. Transformation by isolated L-form DNA was indistinguishable from DNA isolated from bacillary strains and
the transformation efficiency of isolated DNA was not stimulated by the L-form lysate (Table 3). If the physical state of the lysate DNA were responsible for the increased transformation, results similar to those found using the L-form lysate DNA might also be obtained by transformation using a lysate prepared from protoplasts of bacillary cells. Gently lysed protoplasts of B. subtilis, prepared as outlined above, were relatively comparable to L-form lysate DNA in their ability to multiply transform RUB125, as shown in Table 4, although the efficiency of single marker transformation by protoplast lysate DNA was generally one-half to threefourths that obtained with an L-form lysate (data not shown). Unselected marker transformation. The linkage between trpC2 and hisB2 by transformation is normally 50% with isolated DNA (20). Transformation of these markers by the L-form lysate DNA (RUB321 recipient) and by the
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J. BACRIOL.
BETTINGER AND YOUNG TABLE 3. Effect of MA L-form lysate on transformation of BR151 by Isolated DNAa Transformation components added at:"
Met+ transformants
Expt
0 min
1
L-form lysate, isolated DNAC L-form lysate
L-form lysate 2
15 min
30 min
45 min
(%)
BR151 cells
0.17
BR151 cells isolated DNAC BR151 cells, isolated DNAc BR151 cells
0.10
0.52 0.0
L-form lysate, pronase L-form lysate
isolated DNAd
BR151 cells
1.5 1.0
L-form lysate L-form lysate
isolated DNAd
BR151 cells, isolated DNAd BR151 cells BR151 cells, isolated DNAd BR151 cells
0.9 1.1
BR151 cells BR151 cells BR151 cells
2.0 0.0 0.0
L-form lysate, pro-
0.0
nase
isolated DNAd
Pronase L-form lysate
a Competent BR151 (lys-3,trpC2,metB1O) cells and isolated RUB818 DNA (prototrophic) were prepared as described in the text, and transformation was performed as outlined therein. Pronase was used at 0.5 mg/ml final concentration after addition to L-form lysate or isolated DNA in the transformation tube. The DNA in experiment 2 was prepared similarly to experiment 1 except it was concentrated by Aquacide II in dialysis tubing. b The L-form lysate and isolated DNA were added to the transformation tube at the times indicated to effect a pretreatment prior to the addition of competent cells, after which transformation was allowed to proceed for
30 min. c L-form DNA present at 1 Ag/ml, isolated DNA at 2 jig/ml (fmial concentrations after BR151 competent cell
addition). (RUB818 DNAIL-form DNA, 2.) dL-form DNA present at 0.8 sg/ml, isolated DNA at 5 jg/ml (final concentrations after BR151 competent cell addition). (RUB818 DNA/L-form DNA, 6.2.) TABLE 4. Transformability of RUB125 by gently Iysed L-forms or BR151 protoplastsa Relative transformation of RUB125 by donor lysate DNA preparations
Selected marker(s)
Thr His Ura Leu Thr His His Ura Ura Leu Thr His Ura His Ura Leu Thr His Ura Leu
L-form lysate DNA
BRi51 proto-
1.00 1.10 0.83 0.68 0.11 0.08 0.14 0.01 0.01 0.002
1.00 0.63 0.78 0.71 0.10 0.09 0.08 0.02 0.01 0.003
plast lysate DNA
aA culture of competent RUB125 (thr-2, hisAl,
ura-1, leuA8) was transformed either by gently lysed MA L-forms or BR151 protoplasts as outlined in the Methods. The results are shown relative to Thr+ transformants.
protoplast lysate DNA (RUB128 recipient) showed co-transformation of the trpC2 hisB2 markers at higher frequencies, approaching 100% (1; Table 5). This region is expanded in the SB202 strain (aroB2, trpC2, hisB2, tyrAl). When SB202 was transformed using L-form lysate DNA and Aro+ transformants were selected, the other markers were co-transformed in the order: Trp+ (82%), His+ (77%), and Tyr+ (72%) with Aro+ normalized to 100%. These data are relatively similar if isolated DNA is used, only the co-transformation frequencies were lower as predicted. When Tyr+ was selected in a transformation by lysate DNA, the nonselected, co-transformation of any individual marker or all three was greater than 91%. Dilution analysis of multiple marker transformation. Although the frequencies of multiple marker transformations listed in Table 5 (and reference 2) exceed those expected for congression (13), this does not prove that these genes are taken in on the same fragment of transforming DNA. As discussed by Goodgal
TRANSFORMATION BY CRUDE LYSATE DNA
VOL. 122, 1975
TABLE 5. Co-transformation of non-selected markersa
Recipient
Donor
% Nonselected Select- Nonselect- marker trscoed ed maformaker f°tion
makr
for:
L-form lysate DNA RUB128 RUB818 protoplast lysate DNA
Trp His
89
His Trp
94
SB202
Aro Trp
82
RUB321
L-form lysate DNA
HisB TyrA Trp His
77 72 69
Tyr
SB202
L-form isolated DNA
Aro Trp
His Tyr Trp His
65 53 52 48
991
loci are known to exist on the same chromosome; and a mixture of strains RUB129 (hisAl, ura-1, leuA8), RUB130 (thr-2, ura-1, leuA8), and RUB131 (thr-2, hisAl, leuA8) protoplasts, in which the prototrophic traits under investigation are on different chromosomes. The latter three strains (RUB129, RUB130, and RUB131) were protoplasted separately, adjusted to the same OD,,, of the BR151 protoplasts, and mixed in equal volumes. Serial dilutions of the BR151 protoplasts as well as the protoplast mixture were made, the diluted protoplasts were lysed, and competent RUB132 cells were added. Nonprotoplasted cells were selected against by elimination of leucine and methionine from the minimal medium. The results (Fig. 1) show that BR151 protoplasts (Thr+Ura+His+) transform RUB132 for one, two, or three markers at constant ratios, independent of DNA dilution, but that the frequency of multiple marker transformants ob106
Tyr
SB202
L-form lysate DNA
Tyr Aro
98
Trp His
91 95 91
AroTrp His
aTransformation of competent cells was as described in Materials and Methods. Nonselected transformation markers were supplemented for in the initial transformation isolation and detected by subsequent streaking of isolated colonies onto differential media. The Tyr+ transformants were selected on plates supplemented with phenylalanine, tryptophan, and histidine. Shikimic acid was omitted since phenylalanine alone satisfies the aroB2 requirement. The results are expressed as the percent of 200 selected transformants showing the nonselected phenotype.
(14), if multiple marker transformants arise by the integration of the genes from different fragments of DNA, the frequency of multiple transformations will drop off more rapidly than the number of single transformants as a function of DNA dilution. Similarly, if these genes originate on the same strand, multiple transformants should constitute a constant fraction of single marker transformants upon dilution of DNA. To examine this question, strain RUB132 (carrying thr-2, hisAl, ura-1) was transformed for Thr+, Thr+His+, and Thr+His+Ura+ by protoplast lysate DNA. Two protoplast lysate DNA preparations were used: BR151 protoplasts (Iys-3, trpC2, metBlO), in which the
105 z 4U
LOG2 DILUTION
FIG. 1. Dilution analysis of multiple marker transformation. Serial dilutions of protoplast lysate DNA were used to transform cells of RUB132 (thr-2, hisAl, ura-1) with two preparations of DNA as described in the text. Donor DNA from protoplasts of strain BR151 (prototrophic for all selected traits): 0, Thr+; A, Thr+His+; and U, Thr+His+Ura+ transformants. Donor DNA from a mixture of protoplasts of strains RUB129, RUB130, and RUB131 (each selected trait from a different strain): 0, Thr+, A, Thr+His+; and 0, Thr+His+Ura+ transformants.
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BETTINGER AND YOUNG
J. BACTERIOL.
tained using the protoplast mixture drops off cell, since when present at only one-sixth the more rapidly with DNA dilution than does the concentration of isolated DNA it inhibits transfrequency for single marker transformation. formation by that DNA by 50%. It is known that after the gentle lysis of B. DISCUSSION subtilis protoplasts under conditions similar to B. subtilis 168 derivatives are transformed by ours, the chromosome remains intact and atDNA made available by the gentle lysis of tached to the cell membrane (17). We have either a stable L-form (MA) or protoplasts at postulated that the high co-transformation obfrequencies up to 10-fold greater than those served using DNA in the L-form lysate (and obtained with DNA isolated by the more con- protoplasts as well) was due to the uptake of the ventional procedures (2; Tables 4 and 5). The markers from a continuous donor DNA molemethod of DNA isolation used involves removal cule (2). The data in Fig. 1 conclusively prove of the cell wall by lysozyme, osmotic lysis of the our hypothesis. Thr+His+ and Thr+His+Ura+ protoplast, pronase digestion, solubilization of co-transformations of RUB132 (thr-2, hisAl, the cytoplasmic membrane by detergent, and ura-1) by BR151 protoplast lysate DNA (protofinally extraction with phenol. The remaining trophic for all markers transformed) is a conDNA is essentially free of protein and RNA. stant fraction of Thr+ transformants at all Hirokawa (16) reported that the level of trans- dilutions of DNA. These results are predicted if fection of B. subtilis by DNA isolated from all markers are taken up from the same, continbacteriophage 029 could be diminished by treat- uous segment of donor DNA (14). Conversely, ment with pronase, although pronase has no co-transformation of RUB132 for the same such effect on transfection with DNA from marker sets by DNA prepared by gently lysing a bacteriophage 44 (21). In view of this, and since mixture of protoplasts of strains RUB129, the lysate DNA preparations used for transfor- RUB130, and RUB131 (each prototrophic gene mation are essentially untreated, the possibility originates from a separate strain) behaves as arose that the loss of certain components during predicted by congression in that only Thr+ DNA isolation was responsible in some way for transformation is a linear function of dilution, the decreased transformation efficiency seen whereas Thr+His+ and Thr+His+Ura+ transforwith isolated DNA. The L-form lysate DNA, mants are not a constant fraction of the Thr+ however, proved refractory to attempts to di- transformants. When SB202 (aroB2, trpC2, minish its transformation efficiency by either hisBl, tyrAl) was transformed by lysate DNA RNase, pronase, or trypsin (Table 2). Even after and Aro+ transformants were selected, the coextensive DNase treatment the lysate retained transformation frequencies of the other markers significant transforming ability for single (0.1% decreased in the order of their replication. If of control) and double (0.01% of control) mark- these markers are integrated in the order of ers. As noted in the text, DNase had a more their replication as suggested by Erickson and pronounced effect on unlinking markers. The Braun (11), then by selecting SB202 transformsuperior biologic activity of the lysate DNA may ants for the marker last in the replication order reflect a more native physical nature. Snyder the frequency of co-transfer of the unselected and Young (23) reported that DNA still at- preceding markers should be similar and (as tached to membrane fragments prepared from shown in Table 5) they are all greater than 90% lysed protoplasts after rigorous homogenization co-transformed. Although this is a closely transformed at efficiencies two- to three-fold linked region even when transformed by isohigher than if the DNA were isolated by conven- lated DNA (Table 5), there is some indication of tional methods. The membrane fragments to polarity in the entrance and/or integration of which the DNA was attached might afford a these transforming genes. protective structural support. It also seems Transforming DNA is thought to attach to a reasonable to suggest that it is membrane competent cell in an endwise fashion (10) and attached DNA, or a fraction thereof, which in subsequently converted to membrane-bound, lysate preparations is resistant to DNase diges- double-strand fragments of approximately 9 x tion and retains the ability after exposure to 106 daltons by endonuclease (1, 7, 9, 15). AlDNase to transform competent cells. The L- though bound, the double-strand fragment is form lysate was tested for its ability to enhance further broken down into single-strand fragthe transforming efficiency of isolated DNA ments of 2.8 x 106 daltons, which complex with (Table 3). Overall, the data indicate that the the recipient genome and are integrated (8). lysate did not increase the transformation by Assuming that the lysate DNA is attached to isolated DNA. Lysate DNA does, however, seem the competent cell, taken up in a linear fashion, able to bind more effectively to the competent and fragmented as just described for isolated
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TRANSFORMATION BY CRUDE LYSATE DNA
DNA, the high incidence of multiple marker transformations observed using lysate DNA is then perhaps due to an ordered arrangement of membrane-bound, single-strand fragments, following linear uptake of a continuous DNA strand and a polarity of gene integration during chromosome replication (12, 13). ACKNOWLEDGMENTS We thank G. A. Wilson for his helpful discussions during the course of this work. This research was aided by grants from the American Cancer Society (VC 27-J) and from the United States Public Health Service (AI-10141) from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Arwert, F., and G. Venema. 1973. Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Mole. Gen. Genet. 123:185-198. 2. Bettinger, G. E., and F. E. Young. 1973. Transformation of Bacillus subtilis using gently lysed L-forms, a new mapping technique. Biochem. Biophys. Res. Commun.
55:1106-1111. 3. Boylan, R. J., N. H. Mendelson, D. Brooks, and F. E. Young. 1972. Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J. Bacteriol. 110:281-290. 4. Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J.
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