Molec. gen. Genetic. 175, 235 237 (1979) ,~/by Springer-Verlag 1979

Short Communication

Restriction of Plasmid-Mediated Transformation in Bacillus subtilis 168 Teruo Tanaka Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi, Tokyo 194, Japan

Summary. When

plasmids carrying leucine genes of

Bacillus subtilis 168 were isolated from a restriction

and modification deficient ( r - m ) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec + strain (transformation by integration of the donor D N A into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r + or r - phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r m strains should be used for construction of recombinant D N A molecules in B. subtiIis 168.

Host controlled restriction and modification have been found in Bacillus subtilis (Trautner et al., 1974; Shibata and Ando, 1974; Uozumi et al., 1977). B. subtilis ~105C grown on various B. subtilis strains are plated on B. subtilis 168 with an efficiency of 10-1-10-3. In contrast, when the same phage grown on B. subtilis 168 was used to infect B. subtilis N , the efficiency was reduced to an order of 10 . 4 to 10 .5 (Shibata and Ando, 1974; Uozumi et al,, 1977). Unlike d~105C, qb29 and SPP1 grown on B. subtilis N are not affected by the restriction of B. subtilis 168 (Shibata and Ando, 1974). These observations indicate that B. subtiIis 168 cells restrict incoming D N A weakly. Molecular cloning in B. subtilis was effected by the introduction of drug resistance factors of Staphylococcus aureus and of a nutritional marker attached to a B. subtilis plasmid (Ehrlich, 1977; Ehrlich, 1978; Gryczan and Dubnau, 1978; Keggins et al., 1978; Tanaka and Sakaguchi, 1978). F r o m the results using drug resistance factors, it was found that the efficiency of transformation of B. subtilis with drug resistance

factors isolated from S. aureus and B. cereus was between 1-1/50 the efficiency with those isolated from B. subtilis 168 into which the resistance factors had been introduced by transformation (Bernhard et al., 1978; Ehrlich, 1977; Gryczan et al., 1978). The authors (Ehrlich, 1977; Gryczan et al., 1978) attributed the reduced efficiency of S. aureus grown plasmid in transformation to the restriction of B. subtilis 168. Recently an alternative explanation for the difference in transformation frequency described above was presented by Canosi et al. (1978) who found that the specific transformation activity of monomers of S. aureus drug resistance factors was less than one thousandth the activity of the mulimeric plasmid DNA. They also showed that ligation of linearized plasmid D N A by a restriction enzyme gave higher transformation frequency than did the intact monomer molecules. F r o m these observations, they concluded that concatemeric form D N A is responsible for the transformation activity in B. subtilis and suggested that the frequency of oligomeric D N A may be higher in plasmids derived from B. subtilis than in preparations of the same plasmid directly isolated from S. aureus (Canosi et al., 1978). Therefore, a clear answer t o the effect of restriction and modification in B. subtilis 168 on the frequency of the plasmid transformation has not been available. This must be studied under the condition where the molecular structure of a plasmid is neglected. To clarify whether the host controlled restriction and modification affects the transformation efficiency with plasmid DNAs, restriction and modification deficient strains were used in this study and the frequency of transformation in the wild type cell and the mutant cell was compared. Plasmids used were pLS102 and pLS103 in which two and one E c o R I fi'agment carrying B. subtilis leucine genes are linked to a B. subtilis (natto) plasmid, respectively. A preparation of pLS102 from the host

0026-8925/79/0175/0235/$01.00

236

T. Tanaka : Restriction of Plasmid-Mediated Transformation

cells contain pLS103 in addition due to the in vivo recombination of pLS102 (Tanaka and Sakaguchi, 1978). The strains used are listed in Table 1. The r e c E 4 mutation is necessary to avoid integration into the c h r o m o s o m e of a plasmid which carries D N A sequences homologous to those of the recipient chrom o s o m e (Keggins et al., 1978; T a n a k a and Sakaguchi, 1978). When the mixture of pLS102 and PLS103 was isolated from a restriction and modification deficient (rMm~) strain B. subtilis M T 1 2 0 (Table 1) and used + + to transform a rMmu strain M T 128, the transformation efficiency of the plasmids was greatly reduced (Table 2) irrespective of the molecular weight of the plasmids (pLS102, 10.7 x 106 and pLS103, 6.5 x 106). On the other hand, when the plasmids were isolated from M T 128 and used for transformation, approxi+ + mately the same efficiency was observed for rMmM and rMmM strains. Here, the molecular structure of a plasmid which is reported to determine the transformation frequency can be neglected, since the same

plasmid preparations are used to transform rM+mM+ and r ~ m ~ strains. In contrast to the case of recE a strains, the efficiency of transformation of rec + strains was almost the same irrespective of whether the plasmid D N A s were obtained from the rM+mM+ or rMmM cells (Table 3). Extra chromosomal D N A was not detected in the rec ÷ transformants thus obtained (20 colonies were checked), showing that the homologous D N A on the plasmid was integrated into the chromosome. This observation is similar to the results of Trautner et al. (1974) who showed that the restriction and modification in B. subtilis did not affect transformation with bacterial D N A , and suggests that the plasmid D N A is processed like the transforming c h r o m o s o m a l D N A (Dubnau et al., 1973). The results described here are interpreted as that the plasmid transformation in B. subtilis is affected by host controlled restriction and modification. This implies that use of restriction deficient strains is particularly important when recombinant D N A molecules are constructed in B. subtilis. Keggins et al. (1978) have successfully cloned trp genes from other bacterial D N A sources. Presumably the constructed plasmids did not have the D N A sequence recognized by the B. subtilis 168 restriction system. Thus, one should keep in mind that although the restriction by B. subtilis 168 is weak, some D N A is strongly restricted as shown in this paper. In B. subtilis, the transforming D N A is processed during entry into the cell and the single stranded D N A produced in this process is integrated into the c h r o m o s o m a l D N A ( D u b n a u et al., 1973). The transformation using this process is not affected by the restriction and modification as described in this study and in the other paper (Trautner et al., 1974). The same mechanism cannot be applied to the plasmidmediated transformation of the recE4 cells, since the efficiency was greatly reduced by the host controlled restriction. Recently, Contente et al. (1978) using S. aureus drug resistance factors showed that only cova-

Table 1. B. subtihs strains used in this study Strain

Genotype

References

MIll2

leuA8 argl5 thr5 recE4 rMmra

MT127

leuB6 trpC2 rMmg

Tanaka and Sakaguchi (1978) Constructed by transformation of B. subtilis 168 thy trpC2 with DNA from CU134 (Ward and Zahler, 1973)

MT128 MTll9

leuB6 recE4 rumg

MT120

leuB6 recE4 rr~mM

+

+

+

+

leuB6 trpC2 rr~mM

MIll2 DNA~MT127 CU134 DNA ~+RM125 argl 5 leuA8 r~m~ (Uozumi et al., 1977) MIll2 DNA--, MT119

The restricting/modifying phenotype is designated according to Uozumi et al. (1977)

Table 2. Frequency of transformation with plasmids obtained from restricting and nonrestricting strains Recipient

MT120 (rgm~, recE4) MT128(r~m~,recE4)

No. of Leu+ transformants pSL102 & pLS103-MTI20

pLS102 & pLS103-MT128

large colonies

small colonies

large colonies

small colonies

41,500 (1) 25(6x10 4)

166,000 (1) 220(i.3x10 -3)

9,300 (1) 8,800(9.5x10 1)

36,000 (1) 36,000(1)

pLS102 & pLS103-X refers to the plasmids pLS102 and pLS103 obtained from strain X. The cells carrying pLS102 also harbor another plasmid pLS103. When transformed into B. subtilis recE4 cells, pLSI02 and pLS103 give rise to large and small colonies respectively (Tanaka and Sakaguchi, 1978). The parentheses indicate the relative transformation efficaency.DNA concentration was 1 /ag/ml. Transformation was carried out as described previously (Tanaka and Sakaguchi, 1978) except that the phosphate concentration was reduced to 1/10

T. Tanaka : Restriction of Plasmid-Mediated Transformation Table 3. Transformation of rec + strains with plasmids obtained from restricting and nonrestricting strains recipient

MT119 (rMm~) MT127 (r~m~)

No. of Leu + transformants pLS102 & pLS103 •MT120

pLS102 & pLS103 .MT128

5.7 x 104 8.5 × 104

3.1 × 10 4 5.1 × 104

DNA concentration was 0.1 gg/ml

l e n t l y c l o s e d c i r c u l a r D N A h a s a t r a n s f o r m a t i o n activity, a l t h o u g h it is n o t k n o w n w h e t h e r t h e m o l e c u l a r s t r u c t u r e n e c e s s a r y is s i n g l e o r d o u b l e s t r a n d e d D N A . Since transformation o f t h e r e c E 4 cells b y p l a s m i d D N A is s t r o n g l y r e s t r i c t e d as s h o w n h e r e a n d restriction endonucleases are generally specific for double-stranded DNA, I believe that double-stranded D N A e n t e r s a n d t r a n s f o r m s t h e r e c i p i e n t cells.

References Bernhard, K., Schrempf, H., Goebel, W.: Bacteriocin and antibiotic plasmids in Bacillus cereus and Bacillus subtilis. J. Bacteriol. 133, 897 903 (1978) Canosi, U., Morelli, G., Trautner, T.A. : The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis. Mol. Gen. Genet. 166, 259267 (1978) Contente, S., Dnbnau, D. : Characterization of plasmid transformation in Bacillus subtilis: Kinetic properties and the effect of DNA conformation. Mol. Gen. Genet. 167, 251 258 (1979)

237 Dubnau, D., Davidoff-Abelson, R., Cirigliano, C. : Fate of transforming deoxyribonucleic acid after uptake by competent Bacdhts subtilis: Phenotypic characterization of radiation-sensitive recombination-deficient mutants. J. Bacteriol. 113, 273-286 (1973) Ehrlich, S.D. : Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 74, 1680-1682 (1977) Ehrlich, S.D. : DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75, 1433-1436 (1978) Gryczan, T.J., Contente, S., Dubnau, D. : Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol. 134, 318 329 (1978) Gryczan, T.J., Dubnau, D.: Construction and properties of chimeric plasmids in Bacilhts subtilis. Proc. Natl. Acad. Sci. U.S.A. 75, 1428 1432 (1978) Keggins, K.M., Lovett, P.S., Duvall, E.: Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc. Natl. Acad. Sci. U.S.A. 75, 1423-1427 (1978) Shibata, T., Ando, T. : Host controlled modification and restriction in Bacillus subtilis. Mol. Gen. Genet. 131,275 280 (1974) Tanaka, T., Sakaguchi, K. : 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. Mol. Gen. Genet. 165, 269276 (1978) Trautner, T.A., Pawlek, B., Bron, S., Anagnostopoulos, C. : Restriction and modification in B. subtilis. Biological aspects. Mol. Gen. Gent. 131, 181-191 (1974) Uozumi, T., Hoshino, T., Miwa, K., Horinouchi, S., Beppu, T., Arima, K.: Restriction and modification in Bacillus species. Genetic transformation of bacteria with DNA from different species, Part I. Mol. Gen. Genet. 152, 65-69 (1977) Ward, J.B., Zahler, S.A.: Genetic studies of leucine biosynthesis in Bacilhts subtilis. J. Bacteriol. 116, 719-726 (1973) Communicated

b y E. B a u t z

Received May 2, 1979

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Molec. gen. Genetic. 175, 235 237 (1979) ,~/by Springer-Verlag 1979 Short Communication Restriction of Plasmid-Mediated Transformation in Bacillus s...
NAN Sizes 0 Downloads 0 Views