Molec. gen. Genet. 167, 251 258 (1979) © by Springer-Verlag 1979

Characterization of Plasmid Transformation in Bacillus subtilis: Kinetic Properties and the Effect of DNA Conformation* Sara Contente and David Dubnau Department of Microbiology, The Public Health Research Institute of The City of New York, Inc., 455 First Avenue, New York, New York 10016, USA

Summary. T r a n s f o r m a t i o n o f c o m p e t e n t cells o f Bacillus subtilis with a n t i b i o t i c resistance p l a s m i d D N A

has s h o w n t h a t (a) c o m p e t e n c e for p l a s m i d a n d chrom o s o m a l D N A develops with similar kinetics; (b) D N A linearized with a variety o f r e s t r i c t i o n e n d o n u cleases does n o t t r a n s f o r m ; (c) C C C p l a s m i d D N A is i n a c t i v a t e d for t r a n s f o r m a t i o n by a single n i c k ; (d) T 4 ligase restores t r a n s f o r m i n g activity to b o t h n i c k e d a n d linearized D N A ; (e) C C C relaxed D N A is fully active in t r a n s f o r m a t i o n ; (f) the D N A c o n c e n t r a t i o n - d e p e n d e n c e o f p l a s m i d t r a n s f o r m a t i o n is first o r d e r ; a n d (g) p l a s m i d t r a n s f o r m a t i o n p r o c e e d s with a low efficiency, r e q u i r i n g the u p t a k e o f 103 to 104 DNA molecules per transformant. Based on this i n f o r m a t i o n , a m o d e l for the processing of chromosomal, plasmid and transfecting D N A is p r o p o s e d .

Introduction T r a n s f o r m a t i o n by p l a s m i d D N A is an essential step in m o s t m o l e c u l a r c l o n i n g e x p e r i m e n t s (Sinsheimer, 1977). A n u n d e r s t a n d i n g o f the process b y which c o m p e t e n t cells t a k e u p C C C D N A a n d a l l o w the expression a n d r e p l i c a t i o n o f this D N A m i g h t help i m p r o v e the efficiency o f m o l e c u l a r c l o n i n g e x p e r i m e n t s a n d w o u l d c o n t r i b u t e to o u r k n o w l e d g e o f b o t h b a c t e r i a l t r a n s f o r m a t i o n a n d the b i o l o g y o f plasmids. Abbreviations. Chloramphenicol, Cm; erythromycin, Era; kanamy-

cin, Km; streptomycin, Sin; covalently closed circular, CCC; tryptose blood agar base, TBAB; nicking and closing enzyme, NCE * In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.

This r e p o r t describes the t r a n s f o r m a t i o n o f Bacillus subtilis by D N A f r o m several a n t i b i o t i c resistance p l a s m i d s which we have s h o w n to be useful vectors for m o l e c u l a r c l o n i n g e x p e r i m e n t s ( G r y c z a n et al., 1978; G r y c z a n a n d D u b n a u , 1978). T r a n s f o r m a t i o n o f B. subtilis by D N A f r o m three sources has been d e s c r i b e d : f r o m the b a c t e r i a l chrom o s o m e , f r o m b a c t e r i o p h a g e (transfection), a n d f r o m p l a s m i d s (for recent reviews o f the first two processes, see D u b n a u , 1976 a n d T r a u t n e r a n d Spatz, 1973). W e p r o p o s e a m o d e l to explain the differences a n d similarities a m o n g these three types o f t r a n s f o r m a tions b a s e d on their kinetic p r o p e r t i e s a n d rec-dependence.

Materials and Methods Bacterial Strains and Plasmids. The following strains of B. subtilis, all 168 derivates, were used: BD99 (trpC2, thr-5, his-l), BD170 (trpC2, thr-5), and BD204 (hisB2, thy). All of the plasmids used in this study were originally isolated from Staphylococcus aureus and introduced into B. subtilis by transformation (Gryczan et al.,

1978). They are identified in Table 1. pBD 15 is a high copy number (cop-6) mutant of pE194. The introduction of pE 194 from S. aureus to B. sub tilis and the isolation of the cop-6 mutant is described elsewhere (Weisblum et al., 1978). pBD9, a Km r Emr chimera of pUBll0 and pEI94, prepared by ligating the parental plasmids at their single XbaI sites, is described in Gryczan and Dubnau (1978).

Table 1. Plasmids Plasmid

Antibiotic resistance conferred

pC194 pUBll0 pSA2100 pBD15 pBD9

Cm r Km r Cm r Sm~ Em r Km r Em ~

For offprints contact: Dr. David Dubnau

0026-8925/79/0167/0251/$01.60

252

S. Contente and D. D u b n a u : Plasmid Transformation in Bacillus subtilis

Isolation of Plasmid DNA. All of the plasmid D N A used in this report was isolated from B. subtilis strains by the sodium dodecyl sulfate-NaC1 method of Guerry et al. (1973), followed by ethidium bromide-CsC1 centrifugation (Radloff et aI., 1967) as detailed for B. subtilis by Gryczan et al. (1978).

Plasmid Transformation, Preparation of Bacterial DNA and Competent Cells. Transformation was carried out using frozen competent cells prepared as described by D u b n a u and Davidoff-Abelson (1971). Unless otherwise stated, plasmid D N A and competent cultures were incubated at 37 ° C for 20 rain using 5 gg of D N A per nal. The transformation m e d i u m used was as described by D u b n a u and Davidoff-Abelson (1971) except that 1 m M ethylene glycol-bis (fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA, Sigma, Mo.) was added unless otherwise indicated. Competent cells were preincubated for 5 min at 37 ° C in the E G T A transformation medium before the addition of D N A . Selection for antibiotic resistance was carried out by the overlay method in TBAB agar (Difco, Mich.) after 1.5 h delay at 37 ° C to allow for expression. Selective concentrations of antibiotic were: Cm, 5 gg/ml; Em, 5 gg/ml ; Km, 5 gg/ml; Sm, 35 gg/ml. C h r o m o s o m a l D N A was isolated and used for transformation as described by D u b n a u and Davidoff-Abelson (1971).

Preparation of [3H]-Labeled DNA. BD204 ( p U B l l 0 ) and BD204 (pSA2100) were labeled with thymidine (Moravek Biochemicals, Ca.) as described by Davidoff-Abelson and D u b n a u (1973), using 1.8 ~tg thymidine per ml and 10 gCi [3H]methyl-thymidine per ml. Plasmid and c h r o m o s o m a l D N A were prepared and purified as described above.

Assay for [3H]DNA Uptake. Transformation was carried out as described above using [3H]-labeled c h r o m o s o m a l or plasmid D N A at 5 gg/ml. Samples were withdrawn, treated with 50 gg of D N a s e I per ml for 5 min at 37 ° C, chilled, filtered through Uni-Pore Polycarbonate m e m b r a n e s (25 m m , 0.4 g m pore size, Bio-Rad Laboratories) and washed twice with cold Spizizen salts (Anagnostopoulos and Spizizen, 1961). Filters were dried and radioactivity determined in Toluene-PPO (5 g/l).

Preparation of Nicked Plasmid DNA and Ligation. D N A (100 gg/ ml) was incubated in 10 m M Tris, p H 7.5, 2 m M MgClz, 50 gg of bovine plasma albumin per ml, with 0.1 to 5.0 gg of D N a s e I per ml for 20 min at 0 ° C. The reaction was stopped by the addition of E D T A to 10 m M and heating at 6 5 ° C for 5 min. Nicked preparations were ligated with T 4 ligase (Miles Laboratories, Ind., or New England BioLabs, Mass.) according to the procedure of T a n a k a and Weisblum (1975) except that incubation was at 14° C for 24 h.

Preparation and Assay of NCE. N C E was prepared from 30 to 40 ml of fresh, heparinized chicken blood by the procedure of Camerini-Otero and Felsenfeld (1977). D N A to be relaxed was diluted in Buffer A (200 m M NaC1, 20 m M Tris-HC1, pH 8.0, 0.25 m M E D T A , 5% v/v glycerol) of Camerini-Otero and Felsenfeld (1977) to 20 gg/ml and 1 to 10 gl of N C E added. The mixture was incubated at 37 ° C for 30 to 60 min and the reaction stopped by heating at 6 5 ° C for l0 rain in the presence of 1% sodium dodecyl sulfate. D N A was analyzed for superhelicity on agarose gels using Tris-Mg acetate buffer (see below). Sodium dodecyl sulfate was not added to the reaction mixture when D N A was to be used for subsequent transformation.

Greene et al. (1974) was used routinely. For the resolution of species with varying a m o u n t s of superhelical character, Tris-Mg acetate buffer was used (0.4 M Tris.HC1, p H 8.0, 0.01 M EDTA, 0.05 M Na acetate, 0.005 M Mg acetate [Shure and Vinograd, 1976]). Gels were run at 50 V (4.1 V/cm) at room temperature until the tracking dye (60% sucrose [w/v], 0.1 M E D T A , 0.15% bromphenol blue) ran to the edge of the gel (3 to 4 h). For better resolution, some gels were run at 20 V for 16 h. Gels were stained in ethidium bromide (1 gg/ml) for 30 min at room temperature and destained in HzO for 30 to 60 min. Fluorescence was visualized with a long-wave UV light Transilluminator (C-5, Ultraviolet Products Inc., San Gabriel, Ca.) and photographed on Polaroid 665 film through combined K o d a k gel filters 23A and 8.

Restriction Endonuclease Reactions. All of the restriction endonucleases were obtained from New England BioLabs, Mass. Conditions of incubation were as described (Gryczan et al., 1978). Reaction products were routinely analyzed by agarose gel electrophoresis.

Results

Conditions for Optimum Plasmid Transformation Table 2 shows the effect on transformation of a variety of treatments intended to minimize the nucleolytic inactivation of plasmid D N A during incubation with competent cells. Titration with E G T A of the Ca + + (0.5 mM) normally present in the transformation medium markedly improves the frequency of plasmid transformation, t R N A has a slight and variable effect. The omission of Ca + ÷ from the competence medium lowers the competence of the culture slightly and the addition of E G T A (0.1 to 1.0 mM) to such cultures has little or no effect (unpublished). Our routine procedure is, therefore, to prepare competent cultures in medium which contains 0.5 m M CaC12 and to incubate these cells with plasmid D N A in the presence of 1 m M EGTA. Competent B. subtilis is usually prepared by diluting early stationary stage cultures into fresh medium and incubating with aeration for a period of time which varies somewhat with the strain used (AnagnosTable 2. Effect of E G T A and t R N A on plasmid transformation Treatment

Transformants per ml a ( K m ~)

none tRNA EGTA EGTA EGTA EGTA

1.16 1.92 1.91 4.92 2.87 4.69

(200 gg/ml) (0.5 m M ) (1.0 m M ) (2.0 m M ) (1 m M ) + t R N A (100 gg/ml)

x × x x x x

104 104 104 104 104 104

Agarose Gel Electrophoresis. Plasmid D N A was resolved on vertical 0.8 to 1.0% agarose gels by the procedures of Sharp et al. (1973) and Studier (1973). Agarose (LE) was purchased from Seakem Marine Colloid Corp., Portland, Me. The Tris-borate buffer of

a The BDI70 competent cell suspensions contained 5 x 108 v i a ble cells/ml. Transformation with p U B l l 0 D N A was carried out as described in Materials and Methods

S. Contente and D. Dubnau: Plasmid Transformation in Bacillus subtilis

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Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Molec. gen. Genet. 167, 251 258 (1979) © by Springer-Verlag 1979 Characterization of Plasmid Transformation in Bacillus subtilis: Kinetic Properties...
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