Cloning and expression of AatII restriction-modification system in Escherichia coli

Gene 185 (1997) 105–109

Cloning and expression of AatII restriction-modification system in Escherichia coli Donald O. Nwankwo, Robert E. Maunus, Shuang-yong Xu * New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA Received 5 October 1995; accepted 20 February 1996; Received by F. Barany

Abstract The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns. Keywords: Acetobacter aceti; Endonuclease; Methylase; T7 expression vector; Protein purification

1. Introduction Type II restriction endonucleases are invaluable tools for cloning and physical mapping of genes (Roberts and Halford, 1993). These enzymes usually recognize 3–8 bp of DNA in a highly sequence specific manner, and cleave DNA within or outside their recognition sites (Roberts and Macelis, 1996; Szybalski et al., 1991). The cognate methylase recognizes the same sequence as the endonuclease and can modify either cytosine or adenine within the recognition sequence, rendering the site refractory to endonuclease digestion. This methylation modification plays a protective role for the bacterial chromosome (Heitman, 1993; Roberts and Halford, 1993). A large number of R-M systems have been cloned by * Corresponding author. Tel. +1 508 9275054; Fax +1 508 9211350; e-mail: [email protected] Abbreviations: aa, amino acid(s); A., Acetobacter; aatIIM, AatII methylase gene; aatIIR, AatII restriction endonuclease gene; bp, base pair(s); BSA, bovine serum albumin; DTT, dithiothreitol; E., Escherichia; IPTG, isopropyl-b--thiogalactopyranoside; kb kilobase(s) or 1000 bp; kDa, kilodalton(s); Km, kanamycin; m6A, N6methyladenine; M.AatII, AatII methylase; nt, nucleotide(s); ORF, open reading frame; R-M, restriction-modification; PAGE, polyacrylamide gel electrophoresis; R, resistance/resistant.

the methylase selection method (Szomolanyi et al., 1980; Wilson and Murray, 1991) and a few R-M systems were cloned by the phage restriction activity of the cloned endonucleases (Mann et al., 1978). A small number of R-M systems were cloned based on the in vivo SOS induction triggered by methylation dependent restriction systems (McrA, McrBC, Mrr) of Escherichia coli against cloned methylases (Piekarowicz et al., 1991), or SOS induction triggered by the cloned restriction endonucleases (Fomenkov et al., 1994). The AatII restriction endonuclease purified from Acetobacter aceti recognizes the nt sequence 5∞-GACGTC-3∞ and cleaves between the T and C on both strands of DNA to generate a 4-base 3∞ overhang (Sugisaki et al., 1982). We report here the cloning, sequencing, and expression of the AatII R-M system in E. coli. A purification procedure for the recombinant AatII is also described.

2. Experimental and discussion 2.1. Cloning of aatIIM gene BamHI, HindIII, or PstI fragments of A. aceti chromosomal DNA were ligated into BamHI, HindIII, or

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PstI cleaved and CIP treated pBR322 respectively, and the ligated DNA was used to transform E. coli RR1 competent cells. Plasmids were isolated from the amplified cells to form the primary genomic DNA libraries. The plasmids were digested with AatII endonuclease, and surviving circular molecules were recovered by re-transforming RR1 competent cells with the digested DNA. Plasmids resistant to AatII digestion were recovered from the BamHI, HindIII, and PstI libraries. Further deletion analysis and subcloning indicated that the AatII resistance was conferred by modification of AatII sites and not by loss of AatII sites in the vector DNA (data not shown). 2.2. Cloning of aatIIR gene AatII endonuclease activity was not detected in any of the M.AatII + clones isolated from the BamHI, HindIII, and PstI libraries. Restriction mapping of the M.AatII + clone derived from the HindIII library indicated that there were more than 3 kb of DNA upstream from the methylase gene. Restriction mapping of genomic DNA surrounding the aatIIM gene using the methylase DNA probe in Southern blots indicated that a 5.8 kb NdeI fragment should carry the aatIIM gene and some additional genomic DNA downstream from the aatIIM gene to encode the aatIIR gene. An NdeI genomic DNA library was constructed using pUC19 vector and was subjected to the methylase selection procedure. One M.AatII + clone was recovered from the NdeI library. AatII activity was detected in the cell extract of E. coli carrying plasmid pDN6018 (AatII + and M.AatII +, data not shown). About 4×103 units/g wet cells were detected in extracts from cells containing pDN6018, which is about the same level found in the native strain A. aceti.

1980). Although AatII endonuclease and methylase recognize the same sequence, no significant aa sequence similarity was detected among the two proteins (data not shown).

2.4. Comparative sequence analysis Stretches of homology were found between AatII methylase and several other previously reported m6A methylases, including M.DpnII, M.HindIII, M.HinfI, M.HpaI, M.MboI, M.RsrI, using the program Blastx (Alschul et al., 1990). Fig. 2 shows segments of M.AatII aa sequence similarity to M.DpnII and M.HindIII (Lacks et al., 1986; Nwankwo et al., 1994). When nt sequences surrounding the aatIIRM genes were compared to sequences in the GenBank database using program Blastx (Alschul et al., 1990) and Fasta of the Genetics Computer Group (Devereux et al., 1984), it was found that the upstream DNA has 63% identity (in 630 bp overlap) to an Acetobacter plasmid pAH4 (GenBank accession No. D30784) and the predicted aa sequence from the downstream DNA has 32% identity to the mobilization protein MobL in the Thiobacillus ferroxidans plasmid pTF1 (Drolet et al., 1990; Drolet and Lau, 1992), and 33% identity to the E. coli mobilization protein MobA (Derbyshire et al., 1987; Scholz et al., 1989). The E. coli MobA protein promotes the specific transfer of DNA in the presence of conjugative plasmids. The observation that aatIIRM genes are flanked by nt sequences that share similarity with plasmid-associated genes suggests that the AatII R-M system may be located in a plasmid. However, no small plasmid was detected in the total DNA preparation from A. aceti cells (data not shown). If the aatIIRM genes are associated with an extra-chromosomal element, it may be located in a large plasmid.

2.3. Nucleotide sequence analysis of the aatIIRM genes About 3 kb of DNA encoding the aatIIRM genes were sequenced on both strands using subclones and primer walking (Fig. 1). Two large ORFs aligned in the same orientation were identified. The upstream ORF was assigned as the aatIIM gene based on the deletion analysis of pDN6018 and the predicted conserved aa sequence motifs, DPPY and DPFXGSGXT, characteristic of m6A methylases (Hattman et al., 1985; Wilson, 1992). The aatIIM gene is 996 bp long, encoding a 331-aa protein, with a predicted molecular mass of 36.9 kDa. The downstream ORF, assigned as the aatIIR gene, is 1038 bp in length and encodes a 345-aa protein with a molecular mass of 38.9 kDa. The two genes overlap by 4 bases. The aatIIM gene is 49% G+C whereas the R gene is 52% G+C. The G+C content of the host is in the range of 56–60% (Gillis and De Ley,

2.5. Expression of AatII restriction endonuclease in E. coli A PCR DNA fragment carrying the aatIIM gene was inserted into pSYX19 (a pSC101 derivative, KmR) and transformed into a McrBC− Mrr− BL21 derivative ER2169 (lDE3). The aatIIR gene was amplified by PCR using two primers 5∞-CAACATATG (Ndel site) AATCCAGACGAAGTATTTTCA-3∞, 5∞-TCGAGGGTCGAC (Sall site) TTTAGGATTCTGATTGTGGGA-3∞. The PCR product was cleaved with NdeI and SalI and cloned into pSYX22, a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter (provided by W.E. Jack, NEB; Studier and Moffatt, 1986). The ligated DNA was transformed into M.AatII premodified cells ER2169 (lDE3) [pSYX19-AatIIM, pLysS ]. The T7 lysozyme

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Fig. 1. Nucleotide coding sequence and the predicted aa sequence of the AatII R-M system (GenBank accession No. U65398). The two conserved motifs of N6-adenine methylase are italicized/underlined. The nt sequence preceding the AatII R-M system has 62.5% identity in 630 bp overlap to a plasmid, pAH4, found in Acetobacter sp. BPR2001.

expressed from the pLysS inhibits T7 RNA polymerase activity and serves to keep basal level expression low prior to induction. Cells containing these plasmids were induced for AatII endonuclease production by addition of IPTG to a final concentration of 0.5 mM. About 2.5×105 units of the AatII per gram wet cells was detected following 2 h induction. A 5-fold increase in AatII production was achieved when rifampicin (50 mg/ml ) was added to the culture medium 30 min after IPTG induction. Total proteins in the cell extracts of IPTG-induced and non-induced cultures were subjected to electrophoresis on a 10–20% SDS-polyacrylamide gel. The inducible band migrates to a position corresponding to 39 kDa ( Fig. 3, lanes 3 and 4). This is in close agreement with the calculated size of 38.9 kDa.

2.6. Purification of recombinant AatII restriction endonuclease Nine grams of IPTG-induced cells were sonicated in 200 ml of buffer (10 mM Tris-HCl, pH 7.8, 10 mM b-mercaptoethanol ). Cell debris in the crude lysate was removed by centrifugation and the supernatant was applied onto a DEAE Sepharose column (1.5×15 cm). 260 ml of salt gradient (130 ml of low salt and 130 ml of high salt) were applied from 0 to 1 M NaCl, and fractions collected and assayed for AatII endonuclease activity. The fractions with AatII endonuclease activity were pooled and dialyzed against a buffer containing 75 mM NaCl, 10 mM KPO , pH 7, 1 mM DTT, 1 mM 4 EDTA. The combined fractions were then applied onto

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Fig. 2. Amino acid sequence alignments of M.AatII and M.DpnII (GenBank accession No. P09358), M.AatII and M.HindIII (GenBank accession No. P43871). The segment alignments were generated by Blastx of the National Center for Biotechnology Information (NCBI ).

a Heparin Sepharose column (1.5×15 cm) and proteins eluted with a 260 ml gradient of 0–1 M NaCl. The fractions with AatII endonuclease activity were pooled as before, and dialyzed against a 0.15 M NaCl buffer. The solution was next applied to a phosphocellulose column (1.5×15 cm) and 260 ml of gradient from 0.15 to 1 M NaCl was applied. The fractions with AatII endonuclease activity were pooled and dialyzed against a storage buffer (50% glycerol, 50 mM KCl, 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 1 mM DTT, 200 mg/ml BSA). The final yield was 106 units of AatII endonuclease for 9 g of cells. Two microliters of the purified enzyme were analyzed by SDS-PAGE. IPTG-

induced and non-induced cell extracts were also analyzed in the same gel. Fig. 2 ( lane 7) shows that there are two major protein bands, the 68 kDa BSA and the 39 kDa AatII endonuclease protein.

Acknowledgement We thank William E. Jack for providing the T7 expression system, Elisabeth Raleigh for providing strains, Richard Roberts and Ira Schildkraut for advice, Jay Wayne and Jian-ping Xiao for discussions, and

D.O. Nwankwo et al./Gene 185 (1997) 105–109

Fig. 3. SDS-PAGE of cell extracts and the purified AatII endonuclease. Lanes: 1 and 2, 4 and 8 ml of uninduced cell extracts; 3 and 4, 4 and 8 ml of IPTG-induced and rifampicin-treated cell extracts; 5 and 6, 4 and 8 ml of IPTG-induced cell extracts; 7, purified AatII endonuclease plus BSA. Arrow indicates the purified recombinant AatII.

Regina Donoghue for assistance in preparing the manuscript.

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