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© 1996 Oxford University Press 2268-2271

Footnote

Cloning and expression of the Bal I restriction-modification system

Cloning and expression of the Bal I restriction-modification system Harumi Ueno , Ikunoshin Kato and Yoshizumi Ishino*

Biotechnology Research Laboratories, Takara Shuzo Co. Ltd, Seta 3-4-1, Otsu, Shiga 520-21, Japan

Received March 12, 1996; Accepted April 26, 1996 DDBJ accession no. D82028

ABSTRACT

Bal I, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum , recognizes the DNA sequence 5 ' -TGGCCA-3 ' . We cloned the genes encoding the Bal I restriction endonuclease and methyltransferase and expressed them in Escherichia coli . The two genes were aligned tail-to-tail and their termination codons overlapped. Bal I restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da. The amino acid sequence of Bal I methyltransferase is similar to that of other m 6 A MTases, although it has been categorized as a m 5 C methyltransferase. A high expression system for the Bal I restriction endonuclease was constructed in E.coli for the production of large quantities of enzyme.

INTRODUCTION

Restriction-modification (R-M) systems have been found in a wide variety of bacteria and type II restriction endonucleases are important tools in the field of molecular biology ( 1 ). Many genes encoding the enzymes have been cloned as the R-M systems ( 2 ) and expressed in Escherichia coli cells. Brevibacterium albidum is a corynebacteria, which is irregular non-sporing, Gram positive, rod-shaped and widely distributed in nature ( 3 ). The Bal I restriction endonuclease discovered in B.albidum recognizes the sequence 5'-TGGCCA-3' and cleaves DNA, after the second G, to yield blunt ends ( 4 ). Thus it is a useful six base cutter in recombinant DNA experiments. Bal I methyltransferase is known to methylate the first C and produce C 5 -methylcytosine (m 5 C) in the recognition sequence ( 5 ).

In order to purify large quantities of high quality Bal I endonuclease easily, we cloned the gene encoding the Bal I R-M system and constructed a high expression system for this gene. The expression system in E.coli , as well as the organization and structure of the Bal I R-M system, will contribute to the analysis of the structural and functional relationships of these enzymes.

EXPERIMENTAL AND DISCUSSION

Brevibacterium albidum ATCC15831 was grown in LB broth containing 10 g Bacto-tryptone, 5 g yeast extract and 5 g NaCl at 37oC with shaking. Total cellular DNA from B.albidum was prepared by the procedure described previously ( 6 ). High molecular weight genomic DNA was partially digested with Sau 3AI and 3-7 kbp fragments were separated by agarose gel electrophoresis. These DNA fragments were eluted from the gel and ligated into the Bam HI site of pBBB1, which is a vector having three Bal I recognition sequences. Bal I linkers, 5'-TTGGCCAA-3' (Takara Shuzo, Kyoto, Japan), were inserted into pBR322 at the Eco RV site and the Nlu I site for construction of a vector, pBBB1, to use for Bal I methyltransferase screening. This was done because there is only one Bal I site in the original pBR322 ( 7 ) and it is difficult to digest because it is modified by dcm methyltransferase. Bal I methyltransferase activity was screened for by selecting recombinant plasmids resistant to Bal I endonuclease digestion in vitro . The plasmid isolated from a transformant, obtained after Bal I digestion of the DNA library, contained a 5.2 kbp fragment of B.albidum DNA. A restriction map of the DNA fragment was constructed and some containing deletions were made (Fig. 1 ). The Bal I methyltransferase activities of these deletion derivatives were analyzed and the gene encoding methyltransferase was found to be within a 3.5 kbp Sau 3AI- Eco RV fragment. The plasmid containing the 3.5 kbp fragment was designated pBSR1.


Figure 1 . Restriction map of the DNA fragment containing the genes of the Bal I R-M system in pBB5 and for the deletion analysis. pBSR1, 2 and 3 were deletion derivatives of pBB5. Various restriction fragments removed from pBB5 were cloned into the appropriate sites of pBBB1. The resultant plasmids were then tested for resistance to Bal I endonuclease digestion. pBB5 and pBSR1 recovered from E.coli ER1648 transformants were resistant to Bal I endonuclease digestion in vitro . However, Bal I endonuclease activity was not detected in the extracts from E.coli ER1648 containing pBB5 or pBSR1 at this stage. Open arrows indicate the positions of the structural genes of the Bal I R-M and their transcriptional orientation.

The nucleotide sequence of the DNA fragment was determined by the dideoxynucleotide chain termination method essentially as described by Sanger ( 8 ). Nested deletion clones were generated by the procedure of Henikoff ( 9 ). Dideoxynucleotide chain termination products, obtained using Bca polymerase ( 10 ) and fluorescent-labeled primers, were analyzed in an automated DNA sequencer (ABI 377A; Applied Biosystems). The nucleotide sequence data reported in this work have been deposited with the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession no. D82028.

Sequences were analyzed by using the program DNASIS (Hitachi Software Engineering, Yokohama, Japan). The G+C content and the codon usage of the corynebacteria have been studied ( 11 , 12 ). This is the first report on DNA sequences from B.albidum . The G+C content of the 3.2 kbp fragment was 64 mol%, which can be added to the corynebacterial sequence data (46-78 mol%). Two open reading frames (ORFs) were found in the fragment. Conserved motifs for methyltransferases were found in the deduced sequence from one of the two ORFs (280 amino acids) and therefore this gene seems to be the structural gene for Bal I methyltransferase. The other ORF was aligned tail-to-tail with the ORF described above. The termination codons of both genes overlapped (Fig. 1 ). Bal I endonuclease activity could not be detected in an extract prepared from E.coli carrying pBSR1.

Bal I methyltransferase has been described as producing m 5 C in its recognition sequence, without, however, experimental evidence ( 5 ). However, the existence of the conserved motifs I and IV and their sequence, position and order found from our sequence comparison shown in Figure 2 suggest that this Bal I methyltransferase belongs to the m 6 A[alpha] family of N 6 -adenine methyltransferases proposed by Wilson ( 13 ). Therefore, we confirmed the methylation position within the Bal I recognition sequence. DNA fragments containing synthetic DNA, shown in Figure 3 , were inserted into pBSR1. Bal I recognition sequences overlap a Hin dIII site in pBSR1-HBH and a Nco I site in pBSR1-HBN, respectively. If Bal I methyltransferase methylates adenine in the recognition sequence TGGCCA, the Hin dIII endonuclease cannot cut at the site next to the Bal I site. On the other hand, if cytosine is methylated as reported earlier, the modification will effect Nco I digestion. Figure 4 shows that pHBN1 was digested with Nco I at only one position, even though it has two Nco I recognition sequences. pHBH1 was digested into three fragments, which shows that the three Hin dIII recognition sites could all be digested. This experiment supports Bal I methyltransferase modifying cytosine, which affects Nco I digestion at a site next to the Bal I site, even if its amino acid sequence is like that of a m 6 A methyltransferase.


Figure 2 . Comparison of the Bal I methyltransferase sequence with the published sequences of other methyltransferases. Multi-alignment of the amino acid sequences of the m 6 A methyltransferases was carried out using the software program DNASIS. Identical and similar amino acid residues are indicated by red and blue letters, respectively. Motifs I and IV, thought to be the S -adenosylmethionine binding site and active site, respectively, are indicated by green lines.


Figure 3 . Construction of the plasmids for analysis of the Bal I methylation site. Two linkers containing Bal I recognition sequence were synthesized and introduced into the Nhe I and Hin dIII sites of pBR322. The insert-containing plasmids were screened by Hpa I digestion. The 500 bp Ssp I- Bam HI fragment was transferred from pBR-HBH or pBR-HBN into pBSR1, which can produce Bal I methyltransferase in E.coli .


Figure 4 . Analysis of the methylation site of Bal I methyltransferase. The reaction mixture containing 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 1 mM dithiothreitol, 50 mM NaCl, 0.5 [mu]g pBSR1-HBH and 1 U Hin dIII in 30 [mu]l or the mixture containing 10 mM Tris-HCl, pH 8.5, 10 mM MgCl 2 , 1 mM dithiothreitol, 80 mM NaCl, 0.5 [mu]g pBSR1-HBN and 1 U Nco I was incubated at 37oC for 1 h and resolved by agarose gel electrophoresis.


The N-terminal amino acid sequence obtained from purified Bal I endonuclease exactly matched with that deduced from the nucleotide sequence of the other ORF in pBSR1. The molecular weight of the Bal I endonuclease, estimated from SDS-PAGE, also agrees well with its weight predicted from its nucleotide sequence (260 amino acids). Therefore, we concluded that the ORF is the structural gene for the Bal I endonuclease. To construct an expression system for the Bal I restriction endonuclease, the entire structural gene was amplified by PCR using two primers (5'-dATGGATTACGCGTTTCGTGACCGGCCGCTC-3' and 5'-dCTAGTTACGGTCGTCCCAACCGTGCGGCCA-3'). The initiation codon included in the forward primer was converted from GTG to ATG. The amplified DNA fragment was connected to the lac promoter of pSTV28 (Takara Shuzo), which has a P15A origin, and, to co-transform with the Bal I methyltransferase gene in pBSR1, a derivative of the ColE1 plasmid. The resultant plasmid was designated pSBR8. The Bal I endonuclease gene was deleted from pBSR1 by cutting out the Apa I- Bam HI fragment and religating the plasmid. The plasmid containing only the Bal I methyltransferase gene was designated pBSA1. Bal I endonuclease activity was detected in a crude extract prepared from E.coli ER1648 cells carrying pSBR8 and pBSA1. This activity was induced by the addition of isopropyl-[beta]-D-thiogalactoside to the culture, which means that expression of the Bal I endonuclease gene is controlled by the lac promoter in the plasmid (Fig. 5 ). The recombinant E.coli efficiently produced Bal I endonuclease and 70 000 U of activity were obtained from the crude extract of 1 g cells, which is 100-fold higher than that obtained from B.albidum .


Figure 5 . Detection of the Bal I endonuclease in E.coli carrying the MBalI and RBalI genes. Escherichia coli strains were grown at 37oC with shaking in 100 ml L-broth containing 100 [mu]g ampicillin and 30 [mu]g chloramphenicol per ml. When the A 600 of the culture reached 0.6, isopropyl-[beta]-D-thiogalactopyranoside was added to a final concentration of 1 mM and the culture was incubated for a further 5 h. The cells were harvested (1 g) and suspended in 5 ml sonication buffer containing 20 mM Tris-HCl, pH 7.5, 10 mM 2-mercaptoethanol. The cells were then disrupted by sonication and centrifuged at 10 000 g for 10 min. The supernatant was diluted 50-fold and 1 (lanes 2, 5 and 8), 3 (3, 6 and 9) or 5 [mu]l (4, 7 and 10) was added to the Bal I reaction mixture containing 20 mM Tris-HCl, pH 8.5, 7 mM MgCl 2 , 7 mM 2-mercaptoethanol, 0.01% BSA and 0.68 [mu]g [lambda] DNA in a total reaction volume of 30 [mu]l. Three extracts were compared: E.coli ER1648, without any plasmid (lanes 2-4) and ER1648 carrying pBSA1 and pSBR8 without IPTG induction (5-7) and with IPTG induction (8-10). The reactions were carried out for 1 h at 37oC and the products analyzed by 0.7% agarose gel electrophoresis. [lambda] DNA was digested with commercial Bal I and loaded as the marker for the Bal I digestion profile (lane 1).

ACKNOWLEDGEMENT

We are grateful to the manufacturing division of Takara Shuzo for supplying the purified Bal I endonuclease protein for determination of its N-terminal sequence.

REFERENCES

1 Roberts,R.J. and Macelis,D. (1994) Nucleic Acids Res., 22, 3628-3639. MEDLINE Abstract

2 Wilson,G.G. and Murray,N.E. (1991) Annu. Rev. Genet., 25, 585-627. MEDLINE Abstract

3 Jones,D. and Collins,M.D. (1986) In Sneath,P.H.A., Mair,N.S., Sharpe,M.E. and Holt,J.G. (eds), Bergey's Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore, MD, Vol. 2, pp. 1261-1434.

4 Gelinas,R.E., Myers,P.A., Weiss,G.A., Muray,K. and Roberts,R.J. (1977) J. Mol. Biol., 144, 433-440.

5 Gunthert,U. and Trautner,T.A. (1984) Curr. Topics Microbiol. Immunol., 108, 11-22.

6 Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Seidman,J.G., Smith,J.A. and Struhl,K. (1987) Current Protocols in Molecular Biology. John Wiley & Sons, New York, NY, Vol. 1.

7 Watson,N. (1988) Gene, 70, 399-403. MEDLINE Abstract

8 Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. MEDLINE Abstract

9 Henikoff,S. (1984) Gene, 28, 351-359. MEDLINE Abstract

10 Uemori,T., Ishino,Y., Toh,H., Asada,K. and Kato,I. (1993) J. Biochem. (Tokyo), 113, 401-410.

11 Yamada,K. and Komagata,K. (1970) J. Gen. Appl. Microbiol., 16, 215-224.

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13 Wilson,G.G. (1992) Methods Enzymol., 216, 259-279. MEDLINE Abstract

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*To whom correspondence should be addressed at: Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan
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