NIGXI

Molec. gen. Genet. 165, 269-276 (1978)

© by Springer-Verlag 1978

Construction of a Recombinant Plasmid Composed of B. subtilis Leucine Genes and a B. subtilis (natto) Plasmid: Its Use as Cloning Vehicle in B. subtilis 168 Teruo Tanaka and Kenji Sakaguchi Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan

Summary. Recombinant plasmids composed of Bacillus subtiIis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B.leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLSI02, pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign D N A (Escherichia coli plasmid pBR322) into the B a m N I site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS 103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous D N A (pBR322) into this EcoRI site inactivated both leuA and leuC functions.

duction function. However, direct selection of transformants was impossible. Antibiotic resistance specified by plasmids of Staphylococcus aureus (Ehrlich, 1977) and B. cereus (Bernhard et al., 1978) has been established in B. subtilis 168. In these cases, direct selection for transformants is possible but the insertion of drug resistance into B. subtilis might cause concern about possible widespread dissemination of drug resistance among this bacterial species in which no natural drug resistance plasmid has yet been reported. These notions prompted us to construct a recombinant plasmid that can give an easily detectable phenotype to the transformant and is also safer for use in studying cloned DNA. We have already reported that at least seven kinds of cryptic plasmids were found among B. subtilis strains (Tanaka et al., 1977; Tanaka and Koshikawa, 1977). By use of one of these plasmids and the B. subtiIis 168 leucine gene region which had been cloned in E. coli (Nagahari and Sakaguchi, 1978), we succeeded in constructing composite plasmids that permitted the transformed cells to grow in the absence of leucine. This paper describes the construction and characterization of the leucine gene-containing plasmids as well as usefulness of the plasmids as cloning vehicle.

Materials and Methods Materials and Bacterial Strains

Introduction

Developing a molecular cloning system in Bacillus subtilis has become possible after the discovery of plasmids that can function in B. subtilis 168. Lovett et al. (1976) introduced a B. pumilus plasmid into B. subtilis 168 where it expressed its bacteriocin proFor offprints contact." Teruo Tanaka

~H-thymidine (20 Ci/m mol) was purchased from the Radiochemical Center. EcoRI, SmaI and B a m N I restriction endonucleases were prepared as described previously (Tanaka and Weisblum, 1975; Shibata and Ando, 1976). HindIII and HpaI were purchased from Bio Labs (Beverly, MA, U.S.A.). BsuG (Hoshino et al., 1977) was a gift from Dr. T. Uozumi of Tokyo University. T4-phage induced ligase and an R-factor RSF2124 (So et al. 1975) were gifts from F. Hishinuma in our laboratory. RSF2124-B.leu (a recombinant plasmid composed of RSF2124 and the leucine gene region of B. subtilis 168, Nagahari and Sakaguchi, 1978) was a

0026-8925/78/0165/0269/$01.60

270

T. Tanaka and K. Sakaguchi: Construction of Recombinant Plasmids in B. subtilis

gift from K. Nagahari in our laboratory, a¢C-2 phage DNA and a plasmid pLS28 from B. subtilis IAMl114 were prepared as described previously (Tanaka etal., 1977, Tanaka and Koshikawa, 1977). A restriction and modification deficient strain B. subtilis RMI25 r~m~leuA8 argl5 (a derivative of B. subtilis 168) was provided by Dr. T. Uozumi (Uozumi et aI., 1977). B. subtilis RM125 r~m~leuA8 argl5 thr5 recE4 was constructed by transformation of B. subtilis RMI25 with DNA prepared from B. subtilis BD224 thr5 trpC2 recE4 (Dubnau et al., 1973). B. subtilis RM125 r~m~leuC7 recE4 was constructed by sequencial transformation of RM125 r~m~trpC2 argl5 with DNAs from B. subtilis CU7411euC7trpC2 (Ward and Zabler, 1973) and RM125 r~m~leuA8 argl5 thr5 recE4.

Media AT plates consisted of Spizizen's minimal medium (Spizizen, 1958) supplemented with 50 lag/ml each of arginine and threonine and 1.5% agar. AA plates contained, in addition to the ingredients of AT plates, 50 ~tg/ml of the following amino acids; glycine, alanine, valine, isoleucine, phenylalanine, tyrosine, tryptophan, cystein, methionine, proline, aspartic acid, glutamic acid, histidine and lysine. AA medium had the same ingredients as the AA plates except that agar was omitted. For labeling DNA, 4 pCi/ml of 3H-thymidine was added to the AA medium supplemented with 250 gg/ml of deoxyadenosine.

~

10

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50

100

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Fig. 1. Effect of the Mg + + concentration on the frequency of leucine transformation of recE4 and rec + strains of B. subtilis RM125 leuA8. Mg ++ concentration was increased by the addition of MgC12 to Spizizen's minimal medium (Spizizen, 1958). DNA concentrations (pLS102) were 0.2 gg/ml and 0.005 pg/ml for the recE4 ( o - - © ) and rec + ( © - - o ) recipients strains, respectively

pLSI07, an equal amount (0.5 ~tg) of the plasmids was digested with BamNI or EcoRI, ligated and transformed into E. coli C600 (Tanaka and Weisblum, 1975). Transformants were selected for anapicillin resistance and leucine independence.

Transformation Miscellaneous Competent cells were prepared by the medhod of Anagnostopoulos and Spizizen (1961) except that the transfer of the cell suspension from the first growth medium to the second was carried out one hour after the cessation of the logarithmic phase in the first growth medium. Initially, plasmid-mediated transformation of the recE4 strain was not efficient. During attempts to increase the frequency, MgCI2 was found to stimulate the transformation when added to Spizizen's minimal medium (Fig. 1). Eleven fold stimulation was observed at MgC12 concentrations between 40-50 mM for the recE4 strain, while transformation of the rec + strain (transformation by the integration of plasmid DNA into the chromosome) was enhanced 2.8 fold at concentrations between 25-50 mM. Transformation experiments were carried out at a Mg ++ concentration of 40 mM throughout this study. In the case of transformation with DNA eluted from an agarose gel, a 0.7% agarose gel (diameter 7 mm) was cut into 1 mm lengths, each gel section was dipped in 70% ethylalcobol and placed in 0.1 ml of sterile 5M NaC104. After the gel was dissolved at room temperature (after about 30 min), 10 gl portions from each fraction were added to incubation mixtures. Transformants were selected on either AT plates or AA plates.

Construction of Recombinant Plasmids pLS28 and RSF2124-B.leu (0.5 gg each) were digested with EcoRI and treated with T4-induced ligase as described previously (Tanaka and Weisblum, 1975). For the construction of pLSI07, pLSl03 was partially digested with HindIII and treated with the Iigase. The mixture (0.1 ml) was used to transform competent B. subtilis RM125 argl5 leuA8 thr5 r~m~recE4 cells in a total volume of 1.0 ml. 0.1 ml portions of the cell suspension were spread on AT or AA plates and incubated for 3 days. For the construction of chimeric plasmids composed of a E. coli plasmid pBR322 (Boliver et al., 1977) and either pLS103 or

Sucrose density gradient centrifugation, cesium chloride-ethidium bromide (CsC1-EtBr) centrifugation, digestion with restriction endonucleases and agarose gel electrophorsis were performed as described previously (Tanaka et al., 1977). Densitometric tracing of a photo negative was carried out using Zeineh soft laser scanning densitometer (Biomed Instruments, Inc., Chicago, Ill., U.S.A.).

Results Construction of a Recombinant Plasmid Carrying the L e u c i n e G e n e R e g i o n Schematic presentation of the constructed plasmids is g i v e n i n F i g u r e 2. T h e s o u r c e o f t h e l e u c i n e g e n e r e g i o n u s e d in t h i s s t u d y w a s a c o m p o s i t e p l a s m i d c o n s i s t i n g o f E c o R I f r a g m e n t s o f B. s u b t i l i s 168 D N A and of an R-factor RSF2124. This plasmid DNA ( r e f e r e d t o as R S F 2 1 2 4 B . leu) is c a p a b l e o f t r a n s f o r m i n g l e u A 5 , l e u A 8 , l e u B 6 , a n d l e u C 7 m a r k e r s o f B. subtilis 168 ( N a g a h a r i a n d S a k a g u c h i , 1978). A B. s u b t i l i s (natto) p l a s m i d p L S 2 8 ( T a n a k a a n d K o s h i k a w a , 1977) a n d R S F 2 1 2 4 - B . leu w e r e c l e a v e d w i t h E c o R I , l i g a t e d ( T a n a k a a n d W e i s b l u m , 1975) a n d t h e m i x t u r e w a s u s e d t o t r a n s f o r m B. s u b t i l i s R M 1 2 5 a r g l 5 l e u A 8 thr5 rMmMrecE4 cells. T h e r e c E m u t a t i o n w a s a b s o l u t e l y n e c e s s a r y , s i n c e w i t h o u t it t h e l e u c i n e g e n e u s e d w a s integrated into the host chromosomal DNA. After three days of incubation on AT plates two colonies

T. Tanaka and K. Sakaguchi: Construction of Recombinant Plasmids in B. subtilis

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Fig. 2. The structure of the constructed plasmids. N u m b e r s shown denote the molecular weights ( x 10 6) of the D N A fragments. The thick and the thin line indicate the vector and the leucine gene-containing D N A segment respectively. Cleavage sites of E c o R I , B a m N I S i n a i and B s u G are shown around the circles and those of H i n d I I I are presented inside of the circle, Alphabets depict the H i n d I I I fragments shown in Figure 7. Plasmids under the dotted line were constructed in E. coli C600 and the one in the bracket represents the hypothetical structure of pLS202-pLS206 which might have existed before deletion occurred.

appeared. One of the clones was picked and used for further study. W h e n the t r a n s f o r m a n t was grown in A A m e d i u m ( A A m e d i u m was f o u n d to be better for growth of transformants than A T medium) containing 3H-thymidine, and the lysate was centrifuged in a CsC1-EtBr density gradient, a satellite b a n d was observed which comprised 2.4% of the main b a n d D N A (data not shown). The purified plasmid D N A thus obtained was subjected to sucrose density gradient centrifugation with 14C-2 D N A added as a molecular weight marker. As shown in Fig. 3a, two

1b 20 3"0 FRACT ION NUMBER Fig. 3a a n d b. Sucrose gradient centrifugation of 3-H-thymidine labeled plasmid DNA purified by CsC1-EtBr centrifugation. Arrows indicate the position of 14C-2 DNA cosedimented as a molecular weight marker. Sedimentation was from right to left. a CCC DNA obtained from a Leu + transformant b CCC DNA obtained from a small colony that segregated from the Leu ÷ transformant

peaks were observed; the larger one with a S value of 34.6S and the smaller 28.1S. These values correspond to molecular weights of 9.4 x 1 0 6 and 5.9 × 1 0 6 respectively as calculated by H u d s o n ' s equation ( H u d s o n et al., 1968). By agarose gel electrophoresis of the plasmid D N A and by an electronmicroscopic observation, the larger (pLS102) and the smaller plasmid (pLS101) were determined to have molecular weights of 10.7x 106 and 6.5 x 1 0 6 respectively, in agreement with the values estimated by sucrose gradient centrifugation. D u r i n g purification of the t r a n s f o r m a n t on A T plates, two size classes of colonies appeared after 5 days of incubation (when A A plates were used, the same p h e n o m e n o n appeared after 2 days). W h e n one of the large colonies was picked and streaked on plates, again two size classes of colonies appeared (the ratio of the large to the small colonies was 1 to 4.8), indicating that the small colonies segregated f r o m the large colonies. In contrast, small colonies did not give rise to large colonies. The cells of the small colonies h a r b o r e d only one plasmid species (pLS103) sedimenting at a S value of 28.3S (Fig. 3b)

T. Tanaka and K. Sakaguchi: Construction of Recombinant Plasmids in B. subtilis

272

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to an extent o f 1.7% o f the m a i n b a n d D N A in a CsC1-EtBr g r a d i e n t ( d a t a n o t shown). F r o m the extent o f the c o v a l e n t l y closed circular D N A p r e s e n t in the cell a n d the r a t i o o f the q u a n t i t y o f p L S 1 0 2 to t h a t o f p L S 1 0 I (3 : l as c a l c u l a t e d f r o m Fig. 3a), p L S 1 0 2 a n d pLS101 were c a l c u l a t e d to be p r e s e n t at 3.4 a n d 1.8 copies p e r c h r o m o s o m e in the g r o w i n g cell respectively, p L S 1 0 3 was f o u n d to be p r e s e n t at 5.2 copies p e r c h r o m o s o m e ( d a t a n o t shown). G e l e l e c t r o p h o r e s i s o f the p l a s m i d s f r o m the large a n d small c o l o n i e s is s h o w n in F i g u r e 4 a a n d b. pLS103 m o v e d at the same velocity as d i d pLS101.

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10 20 30 Fract ion number Fig. 5a-e. Transforming activity of pLS10I and pLS102 DNA extracted from an agarose gel after electrophoresis, a A gel electrophoretic pattern of pLSI01 and pLS102 prepared from a Leu + transformant. Electrophoresis is from right to left. The open circular (OC) form of pLS101 which should appear between CCC and OC DNA of pLSI02 cannot be observed in the photograph, b A densitometric tracing of the negative of Figure 5a. c Transformation of the leucine marker of B. subtilis RM125 argl5 IeuA8 with DNA eluted from agarose gel slices.

Presence of Leu + Transforming Activity on the Plasmids T o e x a m i n e w h e t h e r the p l a s m i d s i n d e e d c o n t a i n B. subtilis 168 leucine genes, a p l a s m i d p r e p a r a t i o n f r o m a large c o l o n y was e l e c t r o p h o r e s e d in an a g a r o s e gel a n d the D N A was e x t r a c t e d a n d used for t r a n s f o r m a t i o n o f the leuA8 m a r k e r o f B. subtilis R M 1 2 5 . As s h o w n in F i g u r e 5, the p e a k s of t r a n s f o r m i n g activity (Fig. 5c) c o i n c i d e d with the fluorescent b a n d s o f the c o v a l e n t l y closed circular ( C C C ) D N A o f pLS101 a n d pLS102 (Fig. 5a), with a little activity being f o u n d in the o p e n circular region of pLS102. By d e n s i t o m e t r i c analysis o f a negative o f Fig. 5a, the ratio o f the a m o u n t o f C C C D N A of pLS101 to t h a t o f p L S 1 0 2 was e s t i m a t e d to be 1:4.7 (Fig. 5b). This c o r r e s p o n d s to a ratio o f 1:2.8 in the n u m b e r

o f D N A molecules, t a k i n g t h a t the m o l e c u l a r weights o f pLS101 a n d pLS102 are 6.5 x l06 a n d 10.7 x 106 respectively. The p e a k area o f the t r a n s f o r m i n g activity o f p L S 102 was 6.8 times as large as that o f p L S 101, s h o w i n g t h a t the n u m b e r o f molecules o f the two p l a s m i d s does n o t reflect the t r a n s f o r m i n g activity; i.e., pLS102 was 2.4 times m o r e active t h a n pLS101. It was also f o u n d t h a t the fluorescent b a n d o f pLS103 p l a s m i d c o i n c i d e d with the p e a k o f Leu ÷ t r a n s f o r m ing activity ( d a t a n o t shown).

Structure of pLSlO1, p L S I 0 2 and p L S I 0 3 In o r d e r to s t u d y the structure o f pLS101, pLS102 a n d pLS103, the f o r m e r two p l a s m i d s were e x t r a c t e d

T. Tanaka and K. Sakaguchi: Construction of Recombinant Plasmids in B. subtilis

273

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Fig. 6a-v. Cleavage of plasmid D N A with EcoRI, BamNI and HindlIl restriction endonucleases. (a through f) EcoRI digests of a pLS28; b RSF2124-B.Ieu; e pLS102; d pLS101 ; e pLS103; f). DNA; (g through i) BamNI digests of g pLS102; h pLS101 ; i pLS103; j EcoRI digest of 2 D N A ; (k through q) digest with EcoRl and BarnNI of k pLS28 (the arrow indicates the fastest-moving D N A fragment); I pLSI02; m pLS101; n pLS103; o RSF2124-B.leu; p RSF2124; q EcoRI digest of 2 D N A ; (r through t) digests with EcoRI and HindIII of r pLS102; s pLS101; t pLS102; u EcoRI digest of 2 D N A ; v the diagram of cleavage patterns shown in r, s, and t. Digestion with EcoRI and BamNI was carried out as described previously (Tanaka et al., 1977). Simultaneous digestion with EcoRI and HindIII was performed in a incubation mixture described by Smith and Wilcox (1970). Numbers shown denote the molecular weights ( x 10-6) of the D N A fragments. Electrophoresis was carried out in 0.7% (a through q) and 1.5% (r through u) agarose gels

from agarose gels (Tanaka and Weisblum, 1975) and the three plasmids were cleaved with restriction endonucleases. When digested with EcoRI and electrophoresed in agarose gels, the three plasmids gave two D N A fragments, one with a molecular weight of 4.2 x 106 and the other with a molecular weight of 2.3 x 106 (Fig. 6c, d and e). Each of these D N A fragments corresponded to one of the two D N A fragments of pLS28 and RSF2124-B.leu used as starting materials (Fig. 6 a and b respectively). F r o m densitometric quantitation of the bands in Figure 6c, d and e, pLS102 was found to contain 2 moles of the 4.2 x 106- and one mole of the 2.3 x 106-dalton fragments, whereas pLS101 and pLS103 contained one mole each of the two D N A fragments. The sum of the molecular weights of the D N A fragments for each plasmid D N A (10.7 x 106 for pLS102, 6.5 x 106 for pLS101 and pLS103) is in good agreement with the molecular weights of the respective plasmid obtained by agarose gel electrophoresis and electronmicroscopy. Upon digestion with BarnNI endonuclease, pLS102 gave rise to two D N A fragments with molecular weights of 6.5 x 106 and 4.2 x 106 (Fig. 6 g), the sum of which is again in good agreement with the molecular weight of the undigested DNA. pLS101 and pLS103

gave one D N A fragment each with a molecular weight of 6.5 x 106 (Fig. 6h and i). When plasmid D N A preparations were digested with both EcoRI and BamNI, they gave two D N A fragments with molecular weights o f l . 9 x 106 and 2.3 x 106 (Fig. 61, m and n). The D N A bands corresponding to a molecular weight of 2.3 x 106 seemed to comprise two kinds of D N A fragments - i.e., one originating from the vector molecule (Fig. 6k) and the other from the 4.2 x 106-dalton fragment which contains the leucine genes (Fig. 60). This was indeed the case, since the 4.2 × 106-dalton EcoRI fragments of pLS101, pLS102 and pLS103 isolated from gels gave 2.3 x 106- and 1.9 x 106-dalton fragments upon digestion with B a m N I (data not shown). The 2.3 x 106-dalton D N A fragment used as a vehicle (Fig. 6a) was insensitive to B a m N I (compare Fig. 6a and k). Digestion of the three samples pLS101, pLS102 and pLS103 with HindIII and EcoRI gave banding patterns indistinguishable from each other (Fig. 6r, s and t), indicating that the three plasmids are constructed by the same components. In Fig. 2, the structure of pLS101, pLS103 and pLS102 as deduced from the foregoing data is presented. It should be noted that the 4.2 x 106-dalton EcoRI fragments are repeated tandemly in the pLS102 plasmid.

274

T. Tanaka and K. Sakaguchi: Construction of Recombinant Plasmids in B. subtilis

A m o r e d e t a i l e d cleavage m a p o f p L S 103 (pLS 101) was o b t a i n e d as follows ( d a t a n o t shown). Using EcoRI a n d B a m N I sites as reference sites, Sinai, HpaI a n d BsuG sites were m a p p e d . T h e n b y d o u b l e digestion with HindIII a n d one o f those enzymes d e s c r i b e d a b o v e a n d b y c o m p l e t e as well as p a r t i c a l digestion o f purified D N A f r a g m e n t s o b t a i n e d with EcoR1 a n d B a m N I digestion, HindIII cleavage sites were u n a m b i g u o u s l y m a p p e d as shown in F i g u r e 2.

Efficiency o f Transformation with Plasmid D N A s o f Different Size on a recE recipient T r a n s f o r m a t i o n o f a recE strain with purified pLS101 D N A gave o n l y small colonies, w h e r e a s with pLS102 D N A , large colonies were o b t a i n e d p r e d o m i n a n t l y ( T a b l e 1 A). P r e s u m a b l y the small c o l o n i e s w h i c h app e a r e d with p L S I 0 2 D N A were g e n e r a t e d by t r a n s f o r m a t i o n with pLS101 c o n t a m i n a t i n g the pLS102 p r e p a r a t i o n . These results indicate t h a t p L S I 0 2 is responsible for giving rise to the large colonies. In o r d e r to test w h e t h e r the efficiency o f t r a n s f o r m a t i o n d e p e n d s on the m o l e c u l a r weight o f p l a s m i d D N A , t r a n s f o r m a t i o n was c a r r i e d o u t with a p l a s m i d p r e p a r a t i o n c o n t a i n i n g b o t h pLS101 a n d p L S 1 0 2 in a m o l a r ratio o f 1:2.8. As s h o w n in T a b l e 1 B, the n u m b e r o f the small a n d large colonies was a p p r o x i m a t e l y the s a m e i n d i c a t i n g t h a t pLS101 (6.5 x 106 daltons) was 2.8 times m o r e efficient in t r a n s f o r m a t i o n t h a n p L S I 0 2 (10.7 x 106 daltons).

Construction of a Smaller Plasmid (pLSI07) Having one E c o R I Site F r o m the o b s e r v a t i o n t h a t the smaller p l a s m i d gave higher f r e q u e n c y o f t r a n s f o r m a t i o n as d e s c r i b e d a b o v e a n d f r o m the n o t i o n t h a t a p l a s m i d with only one EcoRI site w o u l d be a d v a n t a g e o u s for m o l e c u l a r cloning, pLS103 was next m a d e smaller by p a r t i a l d i g e s t i o n with HindIII f o l l o w e d by ligation a n d transf o r m a t i o n . A t r a n s f o r m a n t h a r b o r i n g a smaller plasm i d t h a n pLS103 was picked. The p l a s m i d ( p L S I 0 7 ) h a d a m o l e c u l a r weight o f 5.5 x 106 a n d was f o u n d to be cleaved once with EcoRI. It has lost HindIII f r a g m e n t s E a n d J (Fig. 7) a n d thus one o f the EcoRI sites. This indicates t h a t the f r a g m e n t E is n o t necessary for the r e p l i c a t i o n f u n c t i o n o f the v e c t o r molecule. A p l a s m i d l a c k i n g the o t h e r EcoRI site has n o t been o b t a i n e d so far.

Table 1. Transformation of leuA8 marker RM125 recE4 with plasmid DNA Plasmid DNA

of

B. subtilis

Concentration No. of transformants/ml Large colonies

Small colonies

< 10 410

630 10

(A) pLS10l pLS102

-

(B) mixture of pLS101 and pLS102

0.2 gg/ml

3.0 x 104

2.9 x 104

0.07 gg/ml

1.7 × 104

1.9 x 10~

In (A), the small (pLS101) and the large (pLS102) plasmid were isolated from 0.7% agarose gel as described previously (Tanaka and Weisblum, 1975). In (B), a plasmid preparation containing both pLS101 and pLSI02 in a molar ratio of 1 to 2.8 was used. Leu + transformants were selected on AA plates after 3 days of incubation at 37° C (large colonies could be scored after overnight incubation). Viable cell count was 5.9 x l0 T per ml

A(I B(I .26) C(I .05)-D(0.91) I E(0.78)/ F(0.44)~ G(0.37).~ H(0.33)---I (0.26).~ J(0.23)--

a b c de Fig. 7a-e. Cleavage of pLS103 and pLS107 with EcoRI and HindlII restriction endonucleases. Digests were electrophoresed in 0.7 (a-e) and in 1.5% (d and e) agarose gels. (a through e) EcoRI digests of apLS103; bpLS107; e2DNA. (d and e) HindIlI digests of d pLS103; e pLS107. Numbers in parentheses indicate the molecular weights (x 10 -6) of HindIII fragments, the sum of which is 7.03 x 1 0 6 daltons

E. coIi C600. R e c o m b i n a n t p l a s m i d s (pLS202-206) Insertion of Foreign D N A into the B a m N I Site of p L S l 0 3 and the E c o R I Site of p L S l 0 7 pLS103 a n d p L S I 0 7 were l i n k e d to the B a m N I a n d EcoRI site o f pR322 respectively a n d a m p l i f i e d in

c o m p o s e d o f p L S 1 0 3 a n d pBR322 c o n t a i n e d o n l y one

B a m N I site. P a r t o f p B R 3 2 2 a n d the region e x t e n d i n g f r o m the B a m N I site to the EcoRI site of pLS103 (clockwise) were deleted (Fig. 2). T r a n s f o r m a t i o n with one o f these p l a s m i d s (pLS206) is shown in Ta-

T. T a n a k a a n d K. S a k a g u c h i : C o n s t r u c t i o n o f R e c o m b i n a n t P l a s m i d s in B. subtilis

T a b l e 2. T r a n s f o r m a t i o n o f / e u A 8 a n d leuC7 m a r k e r s of B. subtiIis R M 1 2 5 recE4 w i t h r e c o m b i n a n t p l a s m i d s DNA

leuA8

leuC7

pLS206 pLS207 pLS208 pLS209 pLS103

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Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

NIGXI Molec. gen. Genet. 165, 269-276 (1978) © by Springer-Verlag 1978 Construction of a Recombinant Plasmid Composed of B. subtilis Leucine Genes...
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