NIGG

Molec. gen. Genet. 151, 203-213 (1977)

© by Springer-Verlag 1977

Transformation in Bacillus subtilis Fate of Transforming DNA in Transformation deficient Mutants

Johannes Buitenwerf and Gerard Venema Department of Genetics, BiologicalCentre, Universityof Groningen, Kerklaan30, Haren (Gn), The Netherlands

Summary. Transformation deficient mutants were isolated by means of selection for sensitivity to methylmethane-sulfonate (MMS). The mutations were introduced into a multiple auxotrophic highly transformable recipient. The transformation deficient strains were characterized with respect to their sensitivity to UV-irradiation and treatment with MMS and mitomycin-C (MC) and with respect to both the physico-chemical and biological properties of reextracted donor DNA. As has been established previously (DavidoffAbelson and Dubnau, 1973b) in the transformation proficient wild-type, double-stranded fragments (DSF), single-stranded fragments (SSF) and donorrecipient complex (DRC) are formed from donor DNA. The mutants we report on are of various types: Mutant 7G-73 (transformation frequency about 25 times lower than in the wild-type) is sensitive to UV-irradiation and treatment with MMS and MC, and is extremely deficient in the production of DRC. Mutant 7G-84 (transformation frequency about 12 times lower than in the wild-type) shows also sensitivity to UV-irradiation and treatment with MMS and MC. However, although it forms DSF, SSF, and DRC, the biological activity of DNA re-extracted from transforming cultures of 7G-84 is much reduced as compared to that of the wild-type. Mutant 7G-97 (transformation frequency about 500 times lower than in the wild-type) shows approximately wild-type resistance to UV-irradiation and treatment with MMS and MC, and forms DSF exclusively; the donor DNA is not processed further. Double mutants 6G-103 and 6G-105, constructed by transformation of mutant 7G-97, with DNA from 7G-84 and 7G-73, are about 1250 and 5000 times less transformable than the wild-type respectively. They are sensitive to UV-irradiation and treatment

with MMS and MC. Mutants 6G-103 and 6G-105 also produce DSF, which are not processed further.

Introduction

In studying the molecular mechanism of recombination, genetic transformation offers certain advantages over other systems of genetic transfer in that the donor DNA can be re-extracted from the recipients and examined both with respect to its physico-chemical aspects and its biological activity (Fox and Hotchkiss, 1960; Venema et al., 1965; Dubnau and DavidoffAbelson, 1971). In Bacillus subtilis, such studies have shown that the donor DNA passes through a phase in which its biological activity is greatly reduced (eclipse phase) and that recombination with the recipient DNA is completed within approximately 20 min at 37° (Venema etal., 1965; Dubnau and Davidoff-Abelson, 1971 ; Arwert and Venema, 1973b). During its initial interaction with the recipient cells the donor DNA becomes fragmented to doublestranded fragments (DSF) which, during transformation, give rise to single-stranded fragments (SSF). These can be isolated if certain precautions are taken (Piechowska and Fox, 1971; Davidoff-Abelson and Dubnau, 1973a). It has been reported that the SSF contribute to the formation of hetero-duplex DNA in which one strand of the recipient DNA has been replaced by a single-stranded segment of donor DNA (Davidoff-Abelson and Dubnau, 1973 b). The sequential transition from one state of donor DNA to the next during transformation suggests that various enzymes are participating in the processing of the DNA. One possible means of further characterizing and defining the individual steps in the transformation process is the isolation of mutants deficient

204

J. Buitenwerf and G. Venema : Transformation in Bacillus subtilis

in transformation. In B. subtilis a number of reports deal with this approach and have, in general, indicated that at least some of the steps postulated are likely to be real (Dubnau et al., 1973 b; Harford et al., 1973; Polsinelli et al., 1973). In a pursuit to further characterize the individual steps in the transformation process and ultimately to identify the enzymes involved, we isolated a number of transformation deficient mutants and have tried to determine which step(s) can not be carried to completion in these mutants. To this aim, reextl"acted donor D N A was characterized both physico-chemically and biologically.

at 37°. Samples were plated on nutrient agar and MMS-sensitive mutants were selected by plating on nutrient agar plates containing MMS (2.5 x 10-3 M; Aldrich-Europe).

Materials and Methods 1

Ultraviolet Irradiation and Host-Cell-Reactivation Samples of exponentially growing cultures in nutrient broth (approximately l0 s cells/ml) were centrifuged and resuspended in the same volume of starvation medium. Samples (5 ml) were irradiated with a Hanovia low-pressure (12-15 W) bactericidal lamp (dose rate 35 erg/mmZ/s) at room temperature in gently agitated petridishes. The colony-forming ability of irradiated cells was determined • by plating on nutrient agar. For testing HCR capacity of the various mutants, a suspension of bacteriophage H1 in nutrient broth was diluted 10-fold with starvation medium, containing dimethylsulfoxide (10% v/v). Portions (3.5 ml) were UV-irradiated and at various times thereafter samples were taken and plated on nutrient agar, both with wildtype and mutant cells.

Strains The B. subtilis strains used in this study are listed in Table 1. A set Of isogenic transformation deficient strains was constructed by transformation of strain 7G-8 with DNA from the original mutants. Transformation for the his or the ade marker and selection for MMS-sensitive his + or ade + transformants was used to transfer the mutation into strain 7G-8. Bacteriophage H1, a virulent bacteriophage of B. subtilis (Arwert and Venema, 1974), was used for testing the host-cell-reactivation (HCR) capacity of the mutants.

Sensitivity to Methyl-methane-sulfonate ( M M S ) and Mitomycin-C ( M C ) Exponentially growing cultures in nutrient broth (approximately 10a cells/ml) were exposed to MMS (7.5 x 10 -3 M) at 37°. Samples were withdrawn as a function of time and appropriate dilutions were plated on nutrient agar. The sensitivity to MC was tested on nutrient agar containing various amounts of MC. The concentration of MC inhibiting growth on plates completely was designated the lethal dose of MC. MC was obtained from Nutritional Biochemical Corporation (Cleveland, Ohio).

Media and Chemicals Unless stated otherwise, all chemicals were obtained from BDH. Minimal medium consisted of Spizizen's (1958) minimal salts plus glucose (0.5%), casein hydrolysate (0.02%, Difco), and growth factors (14 ~tg/ml of each, except for vitamins: 0.4 gg/ml). Starvation medium consisted of minimal salts plus glucose (0.5%). Nutrient broth (2.5%) was obtained from Difco. Minimal agar consisted of Spizizen's minimal salts containing agar (1.5%, Difco) plus the required growth factors. Nutrient agar: nutrient broth (2.5%) containing agar (1.25%, Difco). Mutagenesis Exponentially growing cultures of 8G-5 in minimal medium were exposed to N-methyi-N'-nitro-N-nitrosoguanidine (50p,g/ml, Aldrich Chemicals Co., Inc. Milwaukee) during 90min at 37°. The cells were washed by centrifugation (3 times) with minimal medium and diluted 10-fold in nutrient broth. Segregation was for 7 h 1 Abbreviations used. A~s0=absorbance at 450nm; cpm= counts per min ; DNAase= deoxyribonuclease; DRC = donor-recipient complex; DSF =double-stranded fragments; EDTA= ethylene-diamine-tetra-acetate; HCR=host-cell-reactivation; POPOP=l,4-bis-5-(5-phenyloxazolyl)-benzene; PPO=2,5 diphenyloxazole; SSC=standard-saline-citrate; SSF=single-stranded fragments; MC=mitomycin-C; MMS =methyl-methane-sulfonate; thy, trp, tyr, his, ade, met, nic, ura, rib = genes determining biosynthesis of respectively thymine, tryptophan, tyrosine, histidine, adenine, methionine, nicotinic acid, uracil and riboflavin; Ntr= nontransformable

DNA preparations DNA was isolated using a modification of Kirby's method (1957) according to Venema et al. (1965). Unlabeled transforming DNA was isolated from strain 1G-21. Heavy, radioactive DNA containing (methyl-3H)-thymidine and the heavy isotopes 15N and ZH was isolated from cultures of strain 2G-8. The composition of the medium was as follows: deuteriumoxide (100 ml, Merck); (15NH~)SO ~ (0.2 g, VEB Chemic, Berlin); KzHPO4 (1.4g); KH2PO 4 (0.6g); tri-sodium-citrate (0.1 g); MgSO4 (0.02g); algal deuterated hydrolysate (0.5ml, Merck, Sharp and Dohme); tyrosine (1.4 rag) and (methyl-3H) thymidine (2.5 mCi, specific activity 20.7 mCi/mg, Radiochemical Centre). This medium was inoculated with 1 ml of an overnight culture. To obtain acceptable growth rates of cultures diluted in the medium containing 100% (v/v) deuteriumoxide, the inoculum was first adapted to the heavy medium by growth in the medium described above, except that it contained 50% (v/v) deuteriumoxide. The DNA preparations obtained had a specific activity of 5-10 x 105 cpmAtg DNA. Light, radioactive DNA was obtained from strain 2G-8 grown in minimal medium containing (methyl-3H)-thymidine (1 mCi/ 100 ml, specific activity 20.7 mCi/mg. Radiochemical Centre). The specific activity of the DNA preparations was 1-2 x 105 cpm/lag DNA.

Competence and Transformation Cells were made competent according to a modification of the method described by Bron and Venema (1972). Overnight cultures

205

J. Buitenwerf and G. Venema : Transformation in Bacillus subtilis Table 1. B. subtilis strains used in this study Strain

Genotypic and phenotypic characteristics

1G-21 2G-8 8G-5

tyrl tyrt thy trpC 2 tyr 1 his ade met rib ura nic

7G-8 A7-6

trpC 2 his ade met rib ura nic trpC2 his Ntr-1

7G-73 7G-84 7G-97

trpC 2 ade met rib ura MmsS-1 trpC 2 ade met rib ura Mms~-2 trpC2 his ura Ntr-1

6G-103

trpC2 ura Ntr-1 Mms'-2

6G-105

trpC 2 ura Ntr-1 Mms~-I

Reference and comments

eight-foId auxotrophic, highly transformable strain (Bron and Venema, 1972) tyr + transformant of 8G-5 transformation deficient mutant of SB-25 isolated on DNA plates by M.D. Green MMS sensitive derivative of 7G-8 (this study) MMS sensitive derivative of 7G-8 (this study) transformation deficient derivative o f 7G-8 obtained by transformation with D N A of A7-6 (this study) MMS sensitive derivative of 7G-97 obtained by transformation with DNA of 76-84 (this study) MMS sensitive derivative of 7G-97 obtained by transformation with DNA of 7G-73 (this study)

in minimal medium were diluted to A,50 of 0.5 in minimal medium. After incubation with moderate aeration for 3.5 h at 37 °, the cultures were diluted with an equal volume of starvation medium. The temperature was lowered to 34 ° and the culture was incubated for an additional period of 1.5 h with vigorous aeration, during which period maximal competence developed. Competent cells were transformed with 1.5 ~tg/ml DNA during 45 rain at 34 ° or at 30 °. Further binding of D N A was terminated by the addition of high concentrations of highly polymerised calf thymus D N A and further entry of bounci D N A was prevented by the addition of DNAase I. Transformants were scored on appropriately supplemented minimal agar plates.

Binding, Uptake, and Breakdown o f Exogenous D N A

Binding of D N A is quantitated as the total amount of donor D N A associated with the recipient ceils (this quantity of donor D N A contains both D N A sensitive and D N A resistant to removal with DNAase I). Uptake of D N A is quantitated as the amount of cell associated D N A which is resistant to removal by DNAase I. Binding was essentially measured as described previously (Joenje and Venema, 1975). Competent cells (5 ml) were exposed to 1.5 gg/ml aH-trp+ D N A for 45 min a~: 34 °. Further binding was then stopped by transferring 1 ml of the transforming culture to 1 ml ice-cold starvation medium containing EDTA (4 × D0- 3 M) and 1 mg highly polymerised calf thymus DNA, Samples (1.5 ml) of the suspensions were layered on top of ice-cold stepwise sucrose gradients (6 ml of 5% sucrose on top of 8 ml of 10% sucrose in starvation medium containing EDTA (2 × 10 -2 M) and centrifuged for 15 min at 5000 x g and 4°. After decanting the supernarants and drying the tubes, the pellets were resuspended in starvation medium (1 ml) containing DNAase I (I00 ~tg/ml) and lysozyme (200 gg/ml). After incubation for 30 rain at 37 °, 0.6 mi volumes of lysates were used to determine radioactivity. The uptake o f D N A was measured in the same way, except that prior to the centrifugation step further uptake of D N A was terminated by transferring 1 mI of the transforming culture to 1 ml starvation medium containing DNAase I (80 gg/mI), and incubation for D0 rain at 37 °, Breakdown of transforming DNA was determined by mixing 1 ml o f the transforming culture with 1 ml of ice-cold 6% perchloric acid for 30 min on ice. Subsequently the saz'nples were centrifuged for 30 min at 6000 x g at 4 ° and 0.6 ml of the supernatants were used to determine radioactivity as-described previously (Joenje and Venema, 1975).

Transformation frequencies were determined in the samples in which further entry l~ad been stopped with DNAase L Selection was for trp + transformants.

Physico-chemical Fate of Donor D N A

In the analyses of the physico-chemical fate of donor D N A the general procedures of Piechowska and Fox (1971) and of DavidoffAbelson and Dubnau (1973a) were adapted. a) Preparation of Lysates. Competent cultures (25 ml), supplemented with 40 gg/ml of unlabeled thymine and thymidine 20 min before maximal competence was reached, were exposed to 3H-heavy D N A at 30 °. After 10 rain, further binding of D N A was stopped by the addition of an equal volume starvation medium containing 1 mg/ml highly polymerised calf thymus DNA. At various times afterwards, samples were taken and immediately chilled on frozen solutions containing EDTA (10 -1 M; pH 8.5) and NaC1 (0.15 M). Cells were washed (3 times) by centrifugation and resuspension in the same solution and then concentrated (10fold) in a solution containing EDTA (10 .2 M; pH 6.9), NaCI (0.15 M) and lysozyme (10 mg/mI). After 15 rain on ice and an additional period of 15 min at 37 °, Sarkosyl NL 30 (1%, CibaGeigy) was added. After clearing, the lysates were heated for 20 min at 70 °, pronase (0.5 mg/ml, B-grade Calbiochem, previously autodigested during 2.5 h at 45 °) was added and the lysates were incubated overnight at 37 °. The lysates were dialysed overnight against EDTA (D0-~ M; pH 8.5) + NaCI (0.15 M) and dialysed once more during 2 h against EDTA (I0 3 M; pH 8.5) +NaCI (0.15 M). The dialysed lysates were subjected to CsC1 gradient centrifugation at pH i 1.2. Twenty min after the addition of calf thymus D N A a sample was plated to measure the transformation frequency by selection for trp" transformants. b) Isopycnic Centrifugation. The volume of the lysates was adjusted to 7.26 ml with EDTA (10 _3 M; pH 8.5) + N a C l (0.15 M), and 0.9 ml of K2HPO4 (0.4 M; pH 11.35) were added. The final pH was about l 1.2. Solid CsC1 (Merck Suprapur) was added to adjust the refractive index to 1.4020 (20°). 1~C labeled light B. subtilis D N A (approximately 10 times less as the 3H cpm determined in the lysates) was added as a reference. The solutions (10.2 ml) were centrifuged in polyallomer tubes at 30,000 rpm for 60 h at 20 ° in a 40 fixed-angle rotor of a Spinco L-2 ultracentrifuge.

206 The centrifuge tubes had previously been immersed for 10 s in a solution containing Siliclad (1%, Becton, Dickinson and Company) and dried overnight at 37 °. Fractions (0.3 ml) were collected from the top and diluted with 1.5 ml of 0.1 x SSC. Samples (0.6 ml) of the diluted fractions were used to determine radioactivity.

J. Buitenwerf and G. Venema: Transformation in Bacillus subtilis

c

16ld 3

16 105-

Determination of Radioactivity

Biological Fate of Donor DNA Competent cells (relevant markers trp-tyr +) were transformed with 1.5 gg/ml trp + tyr- DNA. After 10 min at 30 ° further uptake of D N A was stopped by the addition of DNAase I (40 gg/ml). At various times afterwards samples were taken and chilled immediately on frozen starvation medium containing EDTA (10 -2 M). The cells were washed (3 times) by centrifugation with starvation medium containing EDTA (10 2 M) and finally resuspended in SSC containing lysozyme (0.5 mg/ml). After lysis at 37 °, D N A was isolated as described in the section DNA preparations. Biological activities of the re-extracted DNAs were tested on competent 8G-5 cells (relevant markers trp- tyr-) with saturating amounts of transforming D N A according to Arwert and Venema (1973b).

o

:E

c) Sucrose Gradient Centrifugation. Fractions of interest of the CsC1 gradients were pooled, and, if necessary, concentrated against poly-ethylene-glycol 6000 (20%, Merck) in 0.1 x SSC. Samples (0.2 ml) were layered on top of linear (5 to 20%) sucrose gradients, and centrifuged (SW50-1 rotor of a Spinco L-2 ultracentrifuge) at neutral pH for 20rain at 32,500 rpm, and at alkaline pH for 180min at 40,000rpm at 20 °. Fractions (4-drops) were collected from the top and, after the addition of H20 (0.5ml), counted for radioactivity.

Radioactivity was determined with a Nuclear Chicago Scintillation Counter Mark II. Samples (0.6 ml) were added to 10 ml of a mixture containing toluene, Triton X100 (23%, v/v), PPO (0.5%) and dimethyl POPOP (5 x 10-3%). 3H counts were corrected for contaminating t4C counts in the 3H-channel.

100

lUG=

UV-dose (erg/mm 21 Fig. 1. Cell survival as a function of UV-dose. Samples of exponentially growing cells were irradiated with various doses of UV and assayed for colony-forming ability. Because the curves for mutants 7G-73 and 6G-105, and for mutants 7G-84 and 6G-105 were identical, only one curve is presented for each pair of mutants, o - - o - - o : 8G-5; e--e--e: 7G-97; zx--z~--zx: 7G-73 and 6G-105; A - - A - • : 7G-84 and 6G-103

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Results

1. Mutant Purification and Construction of Double Mutants Out of approximately 80 MMS-sensitive mutants obtained from the highly-transformable strain 8G-5, 15 turned out to be deficient in transformation to various degrees. Two of these which showed good binding of transforming D N A were used for further studies. In addition, a transformation deficient mutant isolated as being non-transformable on DNAplates was used (A7-6). A set of isogenic strains was constructed by transformation of strain 7G-8 with D N A from the original mutants. The strains used in this study are listed in Table 1. Because of the relatively low sensitivity to MMS treatment of mutant 7G-97 we were able to construct double mutants by transforming 7G-97 with D N A

id5o

-2 10

10-3 -

i

i°

I

10 2 30 time of MMS-treotment(min) B

Fig. 2. Cell survival as a function of time of exposure to MMS. Samples of exponentially growing cells were exposed for various times to MMS (7.5 x 10- 3 M) and subsequently assayed for colonyforming ability. © - - o ©: 8G-5; e - - o - - o : 7G-97; z~ A - - A : 7 G - 7 3 ; A - - A - - A : 7 G - 8 4 ; ~ [] D:6G-105; I - - I - - I : 6 G - 1 0 3

207

J. Buitenwerf and G. Venema: Transformation in Bacillus subtilis

with the aid of infection with UV-irradiated bacteriophage H 1. The results (not shown) indicated that in neither mutant H C R was impaired, probably indicating that their UV-sensitivity is caused by deficiency in recombination repair. The surviving fraction of bacteriophage H1, irradiated with a UV-dose of 525 erg/mm 2, was about 60% both with the wild-type and the mutant hosts.

Table 2. Sensitivity of the mutants and the wild-type to MC Strain

Lethal MC concentration (gg/ml)

8G-5 7G-73 7G-84 7G-97 6G-I03 6G-105

2.5 x 2.5 x 5.0 x 2.5 x 5.0x 5.0 x

10-2 10- 3 10 -3 10 -2 10 3 10 -a

4. Transformability, DNA Binding, DNA Uptake, and Breakdown of Exogenous DNA from the single mutants 7G-73 and 7G-84 and subsequent selection for MMS-sensitivity.

2. Sensitivity to UV-irradiation, MMS, and MC The sensitivity to UV of the mutants is shown in Figure 1, which demonstrates that mutant 7G-97 is only slightly UV-sensitive. Introduction of the mutations from 7G-73 and 7G-84 in 7G-97 increases its sensitivity to UV to the same level as the original mutants. The same applies to the sensitivity to MMS (Fig. 2) and sensitivity to MC (Table 2).

3. Host-cell-reactivation In order to determine whether the sensitivity to UVirradiation of the mutants (notably that of single mutants 7G-73 and 7G-84 and the double mutants 6G103 and 6G-105) was either caused by a deficient recombination repair system or by a deficient excision repair system, their HCR-capacities were examined

To measure the transformability, the mutants were transformed with 3H-trp+ DNA. After 45 rain at 34 ° binding and uptake of donor DNA were assayed on stepwise sucrose gradients. This method separates unbound DNA (staying behind in the upper part of the gradient) from DNA associated with the recipient cells (Joenje and Venema, 1975). The results are presented in Table 3. Column a of Table 3 shows that all mutants are deficient in transformation, although, among the single mutants 7G-97 is more severely deficient than the other mutants. Transformation in the double mutants 6G-103 and 6G-105 is most severely reduced. In order to determine whether the decrease in transformation capacity of the mutants is caused by poor binding or poor uptake of donor DNA, both the total amount of donor D N A associated with the recipients (binding) and DNAase I resistant association of donor DNA with the recipients (uptake) were determined. The results of these experiments are presented in column b and column c of Table 3 and show that

Table 3. Transformation, and binding, uptake and breakdown of D N A Strain

8G-5 (wt) 7G-73(MmsM) 7G-84(MmsS-2) 7G-97(Ntr-1) 6G-103 (Ntr-1 Mms~-2) 6G-105 (Ntr-1 M m s M )

Transformation frequency (trp+/v.c. x 106)

3H-radioactivity (cpm/v.c. x 106) binding

uptake

breakdown

(trp+/cpm)

(a)

(b)

(c)

(d)

(e)

9900 (100) 357 (4) 780 (8) 20.l (0.2) 8.1 (0.08) 2.2 (0.02)

20.3 11.3 16.4 14.3 20.0 25.1

(100) (56) (81) (70) (98) (124)

10.8 3.3 8.2 0.9 0.6 1.1

(t00) (30) (75) (8) (6) (10)

Spec. transforming activity

70.2 39.8 51.0 15.9 35.4 25.2

(100) (57) (73) (23) (50) (36)

984 (100) 154 (16) 95 (10) 26 (2.8) 14 (1.5) 2.4 (0.3)

(a) DNAase I resistant transformants (trp +) per viable cell (b) Binding of 3H-transforming DNA, that is the total amount of radioactivity associated with recipient cells after 45 min at 34 ° (c) Uptake of 3H-transforming DNA, that is the radioactivity associated with recipient cells in a DNAase I resistant state (d) Breakdown of 3H-transforming D N A measured as acid-soluble counts (e) Specific transforming activity, that is the number of DNAase I resistant transformants per cpm of 3H-transforming D N A retained in a DNAase 1 resistant state The values given are the average of 3-6 experiments. The numbers in between parentheses are percentages of wild-type activity

208 all mutants bind approximately the same amount of D N A as the wild-type. This indicates that the transformation deficiency is not due to deficiency in binding of the donor DNA. In general, however, the uptake of D N A by the mutants is impaired, but the extent of impairment can not account for the observed decrease in transformability as is shown in column e of Table 3, in which the specific transforming activity of the D N A (the number of transformants produced per unit of DNAase I resistant D N A incorporated) is shown. Since it has been suggested that breakdown of D N A in B. subtilis may be an essential requirement for D N A uptake (Joenje and Venema, 1975), we measured the D N A degrading capacities of the mutants. Column d of Table 3 shows that all mutants degrade the donor D N A to acid-soluble products to a somewhat lesser extent than the wild-type. However, the reduction of the D N A degrading capacity is not proportional to the reduction in the transforming capacities of the various mutants.

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5. Physico-chemical Fate of Donor DNA in the Mutants The results presented in the preceding section show that the DNAase I resistant donor D N A in the mutants contributes substantially less to the formation of transformants than the DNAase I resistant donor D N A in the wild-type and that the mutants differ in this respect. In order to establish deviations from the normal processing of donor D N A during transformation, we examined the physico-chemical fate of donor D N A in the mutants. To that purpose the mutants were exposed to 3H-heavy donor D N A for 10 min at 30 °. Further binding of donor D N A was stopped by the addition of excess calf thymus D N A . Samples were taken 10 and 45 min later, and processed as mentioned in Materials and Methods. Figure 3 shows the results. Radioactive donor D N A re-extracted from a 10 min sample of the wild-type banded at the position of DSF, SSF and D R C in CsC1 gradients p H 11.2. For the evidence that the material banding at the position of native D N A consists of donor D N A with a reduced molecular weight see section 6. D o n o r D N A re-extracted after the same interval from mutant 7G-73 banded at the position of DSF and SSF, but at that time no D R C had formed. At the same time D N A re-extracted from mutant 7G-84 banded at the position of DSF and SSF and an additional shoulder in the density profile was found at a position in between D R C and DSF. In comparison with the

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Fig. 3. Distribution of radioactive donor DNA in CsC1 gradients pH 11.2. Competent cells were exposed to SH-heavydonor DNA at 30°. After 10 min further binding was stopped by excess calf thymus DNA, 10 and 5 rain later samples were taken from which lysates were prepared. The lysates were subjected to CsC1gradient centrifugation pH 11.2 during 60 h at 30,000rpm and 20° in a 40 fixed-angle rotor (Spinco). Density increases from the left to the right. The arrows indicate the position of 14C labeled light recipient DNA. In Figure 3A (10 min sample) peaks of DRC, DSF and SSF are visible from the left to the right respectively. Their positions coincide with the positions of double-stranded light and heavy DNA and of single-stranded heavy DNA as determined in a reference gradient. A: 8G-5; B: 7G-73; C: 7G-84; D: 7G-97. The double mutants 6G-103 and 6G-105 showed density profiles similar to that of mutant 7G-97

J. Buitenwerf and G. Venema: Transformation in Bacillus subtilis

209

Table 4. Radioactivity in the various forms of donor DNA as determined from the fractions obtained from CsC1 gradients

The results indicate that mutant 7G-73 does produce DSF and SSF, but is impaired in the production of wild-type like DRC. Both the transition of DSF into SSF and of SSF into the product of intermediate density seem to be impeded. In mutant 7G-84 the formation of DRC is abnormal in the sense that the DRC formed after 10 min does not band at the position of DRC formed by the wild-type after the same time interval. Although perfectly capable of binding DNA (Table 3), the results with mutant 7G-97 show that this mutant is largely deficient to perform the transition of DSF into SSF. Since this transition is intimately connected with uptake (Davidoff-Abelson and Dubnau, 1973 b), this mutant is probably grossly unable to take up DNA. With respect to the physicochemical fate of donor DNA, the double mutants 6G-103 and 6G-105 have the same properties as the single mutant 7G-97. However, the number of transformants they produce is much lower than in the mutant 7G-97 (see Table 3) indicating that the mutations of mutant 7G-73 and mutant 7G-84 concern steps next to the uptake of transforming DNA. In order to facilitate comparison between the wildtype and the mutants, and the various mutants mutually, the amounts of radioactivity associated with the various forms of DNA carrying donor atoms were determined from the CsC1 gradients and are given in Table 4. If the 10 min exposure to donor DNA was terminated with DNAase I instead of with calf thymus DNA, almost no material banding at the position of DSF was detectable (results not shown). This is in agreement with the results obtained by DavidoffAbelson and Dubnau (1973b).

Strain

Time of reextraction (min)

Radioactivity (cpm/ml) DRC

DSF

SSF

8G-5 (vet)

10 45

3050 (34) 4323 (76)

5009 (56) 1157 (20)

922 (10) 198 (4)

70-73 (Mms~-l)

10 45

0 (0) 209 (18)

940 (81) 827 (71)

226 (19) 124 (11)

70-84 (Mms~-2)

10 45

1716 (18) 3543 (55)

6330 (68) 2614 (40)

1287 (14) 300 (5)

70-97 (Ntr-1)

10 45

0 (0) 51 (3)

2079 (94) 1890(91)

142 (6) i31 (6)

60-103 (Ntr-1 Mms~-2)

10 45

0 (0) 45 (2)

2022(94) 2018(91)

139 (6) 150 (7)

60-105 (Ntr-1 MmsM)

10 45

0 (0) 32 (2)

1932(93) 1690 (92)

138 (7) 109 (6)

The radioactivity in the various forms of donor DNA was determined in the fractions of the CsCI gradients shown in Figure 3, and was corrected to the volume of the samples withdrawn from the original culture. The DRC, DSF, and SSF peaks were determined by comparison with a reference gradient containing double-stranded and single-stranded donor DNA and light-14C labeled recipient DNA. The peak of 1"C recipient DNA served as a reference in all gradients and was also used to determine the position of DRC. This implicates that in the column showing the amounts of DRC also mutant DRC is given

position of DRC re-extracted from the wild-type, the DRC of intermediate density was banding at a position 2 or 3 fractions apart from the position of wildtype DRC. Ten minutes after the binding was terminated mutant 7G-97 and the double mutants 6G-103 and 6G-105 showed only DSF, very little, if any SSF and no DRC. After 45 min most of the radioactivity present in DNA re-extracted from the wild-type 8G-5 was in the form of DRC and some material was still present as DSF, but SSF were almost completely absent. After the same time interval mutant 7G-73 had produced a limited amount of material banding at a position in between DRC and'DSF. In comparison with the 10 min sample much donor DNA was still banding at the position of DSF after 45 min, whereas the amount of SSF had somewhat decreased. The density profile obtained with the 45 min sample of reextracted donor DNA from mutant 7G-84 was similar to that of the wild-type 8G-5, except that the relative quantity of DSF was higher than that in the wild-type. In mutant 7G-97 and the double mutants 6G-103 and 6G-105 there was little, if any, further change in the density profiles after 45 min. Only a minute amount of radioactive material was banding at the position of DRC.

6. Sucrose-gradient-centrifugation of DSF Produced by the Mutants In order to compare the size of the DSF produced by the mutants with those produced by the wild-type 8G-5, the fractions of the CsC1 gradients containing DSF were pooled and used for sucrose-gradient-centrifugation. The results at neutral pH are presented in Figure 4 and show that the positions of DSF produced by the mutants are not different from the position of DSF produced by the wild-type 8G-5. This indicates that the mutants do not introduce additional double-stranded breaks in the DSF as compared to the wild-type. On alkaline sucrose gradients there was neither an indication for differences between the rate of sedimentation of denatured DSF produced by the wild-type 8G-5 and the mutants (results not shown). This indicates that the mutants do not introduce additional single-stranded breaks in the DSF as compared to the wild-type.

J. Buitenwerf and G. Venema: Transformation in Bacillus subtilis

210

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Fig. 4. Sucrose gradient centrifugation of DSF. The fractions containing DSF of the CsC1 gradients shown in Figure 3 were pooled and used for sucrose gradient centrifugation. The fractions used of the 10 min samples were fraction 21 from 8G-5; fraction 20 + 21 from 7G-73; fraction 20 from 7G-84 and fraction 2 0 + 2 1 from 7G-97. The fractions used of the 45 min samples were fraction 2 0 + 2 1 from 8G-5; fraction 1 9 + 2 0 + 2 1 from 7G-73; fraction 20 from 7G-84 a n d fraction 19 + 2 0 from 7G-97. Centrifugation was for 120 min at 32,500 rpm at 20 ° in a SW 50-1 rotor (Spinco). Sedimentation was from the left to the right. In the upper and lower row DSF from the 10 min samples and the 45 min samples are shown respectively. The arrows indicate the position of native heavy donor D N A . A: 8G-5; B: 7G-73; C: 7G-84; D: 7G-97. The double m u t a n t s 6G-103 and 6G-105 showed the same sedimentation profile as m u t a n t 7G-97

Fig. 5. Biological activity of re-extracted donor D N A . Competent cells (relevant genotype trp- tyr +) were exposed to trp + tyr- D N A at 30 °. After 10 min further uptake was stopped by D N A a s e I (40 gg/ml). At various times afterwards samples were taken to re-extract D N A . The biological activit~)"of the re-extracted D N A was tested on competent 8G-5 cells (relevant genotype trp- tyr-). The donor marker activity and the recombinant type activity are shown in the left and the right half of the figure, respectively. o - - © - - o : 8G-5 (97x 10-5); e - - e - - e : 7G-73 ( 2 x 10-5); D--D---[]: 7G-84 (12x 10-5). N u m b e r s in between parentheses indicate transformation frequencies

7. Biological Fate of Donor DNA in the Mutants

wild-type was about a factor of 50, the donor marker activities in the two strains in the earliest sample differed about a factor of 300, also indicating that recovery from the eclipse is impeded in mutant 7G-73. The ratio of donor marker activity of the wild-type to that of mutant 7G-73 measured in the 45 rain sample agrees reasonably well with the ratio of the transformation frequencies in the two strains. This also indicates that the interval of 45 min between the addition of DNAase I and the re-extraction of the DNA has been sufficiently long to allow the formation of the maximum level of DRC. The recombinant type activity in DNA re-extracted from mutant 7G-73 after 45 min was extremely low. Although binding and uptake of donor DNA in mutant 7G-84 are approximately the same as in the wild-type, Figure 5 shows that both donor marker and recombinant type activity were much less than in the wild-type. However, in contrast to mutant 7G73, the kinetics of the recovery from the eclipse phase and the increase in recombinant type activity were similar as in the wild-type. This is supported by the

To detect possible differences in the biological fate of donor DNA in the mutants as compared to the wild-type, mutant and wild-type recipients were transformed with trp + tyr- DNA. After 10 min at 30° further uptake of DNA was terminated with DNAase I and at various times afterwards samples were taken and treated as mentioned in Materials and Methods. The preparations of re-extracted DNA were tested for donor marker (trp +) activity, recombinant type (trp + tyr +) activity and for resident marker (tyr +) activity. The donor marker activity and the recombinant type activity were normalized against the resident marker activity. The results (Fig. 5) show that recovery from the eclipse phase of donor marker activity in the wild-type was completed in about 20 min. In contrast, recovery of eclipse of donor marker activity in mutant 7G-73 required a considerably longer time interval than in the wild-type 7G-8. Whereas the difference in transformation frequency between mutant 7G-73 and the

J. Buitenwerfand G. Venema: Transformation in Bacillus subtilis observation that the difference in transformation frequency between mutant 7G-84 and the wild-type (about a factor of 8) is similar to the difference between the donor marker activity measured in D N A re-extracted from mutant 7G-84 and the wild-type (a factor of 10 both for the 2 min and the 45 min) sample. Again this indicates that the maximum amount of D R C has been formed in mutant 7G-84 during this interval. Mutant 7G-97, that showed a level of binding of D N A comparable to the level observed in the wildtype, showed neither donor marker activity nor recombinant type activity in D N A re-extracted after terminating uptake with DNAase I. This agrees with the observation that in D N A re-extracted from mutant 7G-97 no D R C is present (see Fig. 3 and Table 4). The same applies to the double mutants 6G103 and 6G-105. Since donor marker activity and recombinant type activity in D N A re-extracted from samples 45 min after the addition of DNAase I are exclusively associated with DRC (DNA associated with the cells has been removed by the addition of DNAase I), the similarity between the ratios of the transformation frequencies of the mutants and the wild-type, and the ratios of the donor marker activities measured in 45 min samples, indicates that those molecules of D R C that do show biological activity in the mutants contribute to the production of transformants with the same efficiency as in the wild-type.

Discussion

Mutants impaired in transformation have repeatedly been isolated with the aim to characterize the recombination process which is indispensible to transformation. This approach, so far, has met with relatively little success. One of the main difficulties often encountered concerns the inability to locate a defect in the recombination process specifically. In the following we will try to locate the specific deficiencies which result in the decreased transformability of the various mutants presented in this paper. To a large extent mutant 7G-73 is deficient in the formation of D R C ; this follows from the observation that in donor D N A re-extracted 10 min after terminating the binding no material is present banding at the position of DRC, whereas in the wild-type already a considerable amount of such material has been formed in this period. Also after 45 min little material banding at the position of DRC is present. This agrees with the results obtained with experiments in which the biological activity of re-extracted D N A was determined. Only extremely little recombinant type activity could be measured. The results of the

211 isopycnic centrifugation of D N A re-extracted from a sample after 45 min, shows that a small amount of material banding at a density less than that of DSF is formed. It is known that during isopycnic centrifugation re-extracted donor D N A can become located at positions less dense than those corresponding to its physico-chemical state because of complex formation with cellular constituents like proteins or membranous material (Piechowska and Fox, 1971). However, in the present case it is unlikely that the aberrant banding of the D N A is due to complexing of DSF or SSF to such constituents, because the treatment of lysates was such that contaminating constituents were released from the DNA. This treatment has been effective as is shown by the position of radioactive material re-extracted from the wild-type that bands at the position of DSF and SSF. One of the possibilities which may account for the aberrant banding of some of the radioactive material re-extracted from mutant 7G-73 is that this material represents a complex of donor and recipient D N A in which single-stranded material continues to be present. In transformation of the wild-type it has been shown that SSF contribute to D R C (Davidoff-Abelson and Dubnau, 1973b). It is conceivable that the aberrant or unfinished complex from mutant 7G-73 still carries a certain amount of single-stranded donor DNA. With respect to the transition of DSF into SSF, and of SSF into DRC, mutant 7G-73 also deviates from the wild-type. Whereas in the wild-type during a 35 rain period of incubation after re-extraction of the 10 rain sample the decrease of DSF amounts to a factor of 2.8 (56/20, see Table 4), in the same period the corresponding value for mutant 7G-73 amounts to a factor of 1.1 (81/71, see Table 4). This indicates that, as compared to the wild-type, DSF are less efficiently converted into SSF. A similar calculation shows that the same applies to the conversion of SSF into DRC. Because 1) the DRC formed in the mutant is deviating from wild-type D R C and 2) the transition of SSF into mutant DRC is impeded, we tend to assume that mutant 7G-73 is impaired in the integration step of the transformation process. This is substantiated by the observation that mutant 7G-73 is H C R proficient, indicating that the sensitivity to UV-irradiation is in all likelyhood due to a deficient recombination repair system. An alternative explanation for the obviously pleiotropic effect of the mutation (both affecting the D R C formation and the transition of DSF into SSF), is, that recombination and uptake of D N A are intimately associated processes, which are possibly membrane located (Dooley and Nester, 1973). Mutants with a phenotype similar to the one de-

212

J. Buitenwerfand G. Venema: Transformation in Bacillus subtilis

scribed for mutant 7G-73 have been described by Dubnau et al. (1973b). Since we have not carried out a genetic analysis in order to locate the mutation we are not in a position to determine whether this mutant is identical to the mutants of class I or class IIb carrying the recA, the recD or the recE mutation (Dubnau and Cirigliano, 1974). Only a genetic analysis can provide a definite answer with respect to the identity of mutant 7G-73. Because of the absence of a number of data on the physico-chemical fate of donor D N A in the existing literature, which concern: 1) the presence or absence of D R C in samples withdrawn early after terminating binding, 2) the density of the D R C 45 min after terminating binding, 3) the transition of DSF into SSF and of SSF into DRC, and 4) the molecular weight of native and denatured DSF in comparison with the added donor DNA, a phenotypic comparison between mutant 7G73 and presumably comparable mutants is hampered. For similar reasons we are unable to fit mutant 7G-73 in the system described by Mazza et al. (1975). Mutant 7G-84 clearly shows the production of material banding at the position of wild-type DRC, 45 min after binding is stopped, in approximately the same quantity as the wild-type. However, in contrast to the wild-type 10 min after binding has been terminated material with a density slightly higher than the density of wild-type D R C is present in re-extracted DNA. The relative amount of radioactivity in the mutant DRC is substantially lower than the relative amount of radioactivity in D R C present in the wildtype at the same time (see Table 4), indicating that in this mutant restraints are present on the formation of DRC. The same applies to the transition of DSF into SSF. The density of the mutant D R C found in the 10 rain sample, being slightly higher than that of wildtype DRC, could possibly be explained by postulating an intermediate in the transformation process in which single-stranded-ness of the donor moiety exists. Evidence for such an intermediate is presently accumulating (Buitenwerf and Venema, in preparation). The observation that in D N A re-extracted 45 min after the addition of calf thymus D N A D R C is banding at the normal position might be explained by assuming that in mutant 7G-84 the possible intermediate is more slowly converted into D R C than in the wild-type. Arguments against possible complex formation between donor D N A and cellular constituents as an explanation for the presence of aberrant D R C in a 10 min sample, are the same as given for mutant 7G-73. Although after 45 min mutant 7G-84 has formed apparently normal DRC, in quantities comparable to the wild-type, the biological activity measured in D N A re-extracted from this mutant is much lower

than the activity measured in D N A re-extracted from the wild-type. This indicates that D R C molecules formed by mutant 7G-84 do not have the same probability as the D R C molecules formed by the wild-type to yield a viable transformant. As in mutant 7G-73, the mutation transferred into strain 7G-84 has pleiotropic effects. This can most simply be explained by assuming that uptake and integration of donor D N A are integrated processes. In view of the observation that initially mutant D R C bands at an abnormal position in CsC1 gradients and that D N A re-extracted from a 45 min sample shows reduced biological activity, although D R C then bands at the position of wild-type DRC, we hypothesize that this mutation affects the recombination part of the integrated process. This hypothesis is supported by the observation that mutant 7G-84 is H C R proficient, which indicates that its sensitivity to UV-irradiation is in all likelyhood due to a deficient recombination repair system. Alternative explanations for the effects of the mutation in 7G-84 are: 1) the possibility that some of the DRC molecules are degraded afterwards, and 2)the possibility that the formation of D R C is not complete with respect to covalent sealing of donor D N A and recipient D N A (Arwert and Venema, 1973 a; Dubnau and Cirigliano, 1973 a). Comparison between mutant 7G-84 and other transformation deficient mutants of B. subtilis are hampered for the same reasons as mentioned for mutant 7G-73. As compared to the classification system described by Dubnau et al. (1973 b) we suggest mutant 7G-84 to belong to class I or IIa of transformation deficient mutants because, although DRC formation is observed in the same quantity as in the wild-type the transformability of mutant 7G-84 is less than in the wild-type. In mutant 7G-97 almost exclusively DSF and almost no SSF are observed in re-extracted DNA. This indicates that mutant 7G-97 is very much deficient in the conversion of DSF into SSF. As is apparent from Table 4 the small amount of SSF formed is not further processed into DRC. Presently the most acceptable explanation for the severe deficiency of mutant 7G-97 to process the donor D N A further than DSF, is that this mutant is largely deficient in a nuclease which carries the transforming D N A accross the cellular membrane. Lacks et al. (1974) showed that in D. pneurnoniae such a nuclease performs the conversion of double-stranded D N A into DNAase I resistant DNA. In B. subtilis mutants with phenotypes similar to that of 7G-97 can not be fitted in the classification system of Dubnau et al. (1973b), because all the mutants described are sensitive to uV-irradiation and treatment with MMS and MC, whereas mutant 7G-97 is resistant.

J. Buitenwerf and G. Venema: Transformation in Bacillus subtilis

Possibly the mutant is similar to one of the mutants described by Polsinelli et al. (1973) and by Harford et al. (1973). However, because the essential data about binding of donor D N A in those mutants are lacking, no definite answer can be provided whether those mutants have the same characteristics as mutant 7G-97. The density profiles obtained of D N A re-extracted from the double mutants 6G-103 and 6G-105 are almost identical to those obtained from mutant 7G-97. As is shown by Table 4 the amounts of radioactivity present in the various forms of D N A re-extracted from these mutants is closely similar to those determined for mutant 7G-97. Although this might suggest that the introduction of the mutations from 7G-73 and 7G-84 into 7G-97 does not have a detectable effect, the data from Table 3 indicate that the residual transformability of mutant 7G-97 is further decreased. The decrease amounts up to a factor of 3 upon introduction of the mutation from strain 7G-84 and a factor of 10 upon introduction of the mutation from strain 7G-73 into 7G-97. Clearly, the effect of the mutation introduced into mutant 7G-97 is more pronounced in a biological assay system than in the physico-chemical analysis of re-extracted DNA. The transformability decreasing effect of the introduction of the mutations from 7G-73 and 7G-84 into 7G-97 should preferably be ascribed to interference with steps in the transformation process occurring after uptake (most probably the formation of DRC), because no discernable effect is found on the amount of DSF and SSF present in D N A re-extracted from the double mutants as compared to the single mutant 7G-97. This reinforces our conclusion that the defects in mutant 7G-73 and 7G-84 should preferably be located in the process of integration of donor D N A into the recipient genome. Acknowledgements. We acknowledge the excellent technical assistence of Liesbeth Steendam and Fien Helmers. We thank Dr. M.D. Green for putting strain A7-6 to our disposal and Dr. S. Bron and Prof. W.J. Feenstra for critical reading the manuscript. This work was carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid of the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).

References Arwert, F., Venema, G.: Evidence for a non-covalently bounded intermediate in recombination during transformation of Bacillus subtilis. In: Bacterial transformation (L. Archer, ed.), pp. 203-214. London: Academic Press 1973a Arwert, F., Venema, G. : Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA. Molec. gen. Genet. 123, 185 198 (1973b) Arwert, F., Venema, G. : Transfection of Bacillus subtilis with bacteriophage H1 DNA: Fate of transfecting DNA and transfec-

213 tlon enhancement in B. subtilis uvr + and uvr- strains. Molec. gen. Genet. 128, 55-72 (1974) Bron, S., Venema, G. : Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultravioletsensitive derivatives. Mutation Res. 15, 1 10 (1972) Davidoff-Abelson, R., Dubnau, D. : Conditions affecting the isolation from transformed cells of Bacillus subtilis of high-molecular-weight single-stranded deoxyribonucleic acid of donor origin. J. Bact. 116, 146 153 (1973a) Davidoff-Abelson, R., Dubnau, D.: Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis. J. Bact. 116, 154-162 (1973b) Dooley, D.C., Nester, E.W.: Deoxyribonucleic acid-membrane complexes in the Bacillus subtilis transformation system. J. Bact. 114, 711-722 (1973) Dubnau, D., Cirigliano, C. : Fate of transforming DNA following uptake by competent Bacillus subtilis VI. Non-covalent association of donor and recipient DNA. Molec. gen. Genet. 120, 101-106 (1973a) Dubnau, D., Cirigliano, C. : Genetic characterization of recombination deficient mutants of Bacillus subtilis. J. Bact. 117, 488-493 (1974) Dubnau, D., Davidoff-Abelson, R.: Fate of transforming DNA following uptake by competent Bacillus subtilis I. Formation and properties of the donor-recipient complex. J. molec. Biol. 56, 209 221 (1971) Dubnau, D., Davidoff-Abelson, R., Scher, B., Cirigliano, C. : Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtiIis: phenotypic characterization of radiationsensitive recombination-deficientmutants. J. Bact. 114, 273~86 (1973a) Fox, M.S., Hotchkiss, R.D. : Fate of transforming deoxyribonucleate following fixation by transformable bacteria I. Nature (Lond.) 187, 1002-1006 (1960) Harford, N., Samojlenko, I., Mergeay, M. : Isolation and characterization of recombination defective mutants of Bacillus subtilis. In: Bacterial transformation (L. Archer, ed.), pp. 241-267. London: Academic Press 1973 Joenje, H., Venema, G. : Different nuclease activities in competent and noncompetent Bacillus subtilis. J. Bact. 122, 25-33 (1975) Kirby, K.S. : A new method for the isolation of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acids and protein. Biochem. J. 66, 495-504 (1957) Lacks, S., Greenburg, B., Neuburger, M. : Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae. Proc. nat. Acad. Sci. (Wash.) 71, 2305-2309 (1974) Mazza, C., Fortunato, A., Ferrari, E., Canosi, U., Falaschi, A., Polsinelli, M.: Genetic and enzymic studies on the recombination process in Bacillus subtilis. Molec. gen. Genet. 136, 9 30 (1975) Piechowska, M., Fox, M.S. : Fate of transforming deoxyribonucleate in Bacillus subtilis. J. Bact. 108, 680-689 (1971) Polsinelli, M., Mazza, G., Canosi, U., Falaschi, A.: Genetical and biochemical characterization of Bacillus subtilis mutants altered in transformation. In: Bacterial transformation (L. Archer, ed.), pp. 2744. London: Academic Press 1973 Spizizen, J.: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. nat. Acad. Sci. (Wash.) 44, 1072 1078 (1958) Venema, G., Pritchard, R.H., Venema-Schr6der, T. : Fate of transforming deoxyribonucleic acid in Bacillus subtilis. J. Bact. 89, 1250 1255 (1965)

Communicated by W. Arber Received November 9, I976