(D" 1992 Oxford University Press

Nucleic Acids Research, Vol. 20, No. 4 705- 709

Cloning and expression of the Hpal restriction-modification genes Hiroyuki Ito, Harumi Shimato, Atsuko Sadaoka, Hirokazu Kotani, Fusao Kimizuka and Ikunoshin Kato1 Bioproducts Development Center and 'Biotechnology Research Laboratories, Takara Shuzo Co., Ltd, Seta 3-4-1, Otsu, Shiga 520-21, Japan Received December 5, 1991; Revised and Accepted January 28, 1992

ABSTRACT The genes from Haemophilus parainfluenzae encoding the Hpal restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the Hpal endonuclease (R Hpal) to have 254 amino acid residues (Mr 29,630) and the Hpal methyltransferase (M Hpal) to have 314 amino acid residues (37,390). The R Hpal and M Hpal genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coil HB101-HPA2, overproduced R4Hpal. R Hpal activity from the clone was 100-fold that from H. parainfluenzae. The amino acid sequence of M *Hpal was compared with those of other type 11 methyltransferases. INTRODUCTION Various type II restriction-modification systems have been discovered in a wide variety of microorganisms (1). The genes encoding some of these have already been cloned and expressed in Escherichia coli (2- 8). Haemophilus parainfluenzae has two restriction-modification systems, named the HpaI and Hpal systems (9). Of the each gene coding for these systems, only that for HpaH methyltransferase (10) has been already cloned in E. coli. HpaI endonuclease (R* HpaI) recognizes and digests double-stranded DNA at the palindromic sequence 5'-GTTI AAC-3'. HpaI methyltransferase (M HpaI) recognizes the same sequence and methylates the external adenine residue at the sequence 5'-GTTA*AC-3'. M HpaI and R HpaI have been purified by O.J.Yoo et al (11). and DeFilippes (12), respectively. The HincH endonuclease (13) recognizes the sequence 5'-GTPyPuAC-3' and digests also the recognition sequence of the HpaI system. The HincH methyltransferase (M HincDJ) methylates the same adenine residue, as M * HpaI does. Therefore, the R -HpaI gene can be cloned in E. coli cells carrying the M HincIl gene. R * HpaI is one of the most widely used restriction endonucleases in molecular biology. Cloning and expression of the R * HpaI gene in E. coli system will enable to obtain purely the important enzyme in large quantities. In this paper, we report the cloning of the HpaI restriction-modification genes, the cloning

DDBJ accession no. D01166

of the R * HpaI gene with the HincdI system, and the expression of R HpaI. The amino acid sequence deduced for the HpaI methyltransferase was compared with that of other type II methyltransferases.

MATERIALS AND METHODS Bacterial Strains and Plasmids H. parainfluenzae (9) was obtained from the Department of Science, Kyoto University (Japan). E. coli strains HB1I1 [F-, hsdS20(rB-,mB-), supE44, aral4, X-, galK2, lacYl, proA2, rpsL20, xyl-5, md-i, recA13 (14)], RR1 [same as HB1I1 except for recA+ (15)], and JM109 [recAl, endAl, gyrA96, thi,

hsdRJ7(rK-,mK-), supE44, relAl, X-, A(lac-proAB), F',

traD36, proAB, lacJqZAMJ5 (16)] and plasmids pBR322 [Tcr, Apr (15,17)], pACYC184 [Cmr, Tcr (18)], pUC18 [Apr (16)], pPL-lambda [Apr (19)(Pharmacia)], pKHl [Apr (20)], and pNT203 [Tcr (21)] were the host-vector systems used for cloning and expression. M13mpl8 and mpl9 (16) were used for DNA sequencing. E. coli competent cells were produced by the procedure of Hanahan (22).

Enzymes and chemicals Restriction endonucleases and 7-deaza sequencing, kilo-sequence deletion, Megalabel, and ligation kits were products of Takara Shuzo Co., Ltd., and were used according to the manufacturer's instructions. [a-35S]dCTP (37 TBq/mmol) and [Fy-32P]ATP (185 TBq/mmol) were purchased from Amersham International.

Phage and DNA preparation Virulent X phage (Xgt * Xc) was prepared from plate lysates (23). E. coli plasmid DNA was prepared on a small scale by a modification of the method of Bimboim and Doly (24). H. parainfluenzae chromosomal DNA was purified by the procedure of Ausubel et al. (25) Analysis of N-terminal amino acid sequence R HpaI was purified from H. parainfluenzae culture by the procedure of DeFilippes (12). The N-tenninal of purified R - HpaI was sequenced as described elsewhere (13, 26).

706 Nucleic Acids Research, Vol. 20, No. 4

Southern hybridization The genes encoding the HpaI system was isolated by Southern hybridization (27) as reported before (13). A synthetic probe (0.5 jg) was phosphorylated with 1.85 MBq of ['y-32P]ATP using of a Megalabel kit.

Assay of enzyme activities R HpaI activity in vivo was estimated from the plating efficiency of virulent X phage. This activity was assayed in vitro as follows. The enzyme solution was added to the reaction mixture, 25 ,ul of 20 mM Tris-HCl (pH 7.5) containing 10 mM MgC92, 100 mM KCl, and 0.5 Itg of X phage DNA, and incubated at 37°C. One unit of enzyme activity was defined as the amount of enzyme required for complete digestion of 1 ,tg of X DNA at 37°C for 1 hr. M * HpaI activity in vivo was estimated from non-digested plasmids by R HpaI. This activity was assayed in vitro by incubating 10 1l of 50 mM Tris-HCl (pH 7.5) containing 5 mM 2-mercaptoethanol, 10 mM EDTA, 1 ,ag of X phage DNA, 80 AM S-adenosylmethionine, and the enzyme at various concentrations for 1 hr at 37°C. Thereafter 40 jl of a reaction mixture containing R * HpaI was added and the new mixture was incubated at 37°C until digestion was complete. One unit of enzyme activity was defined as the amount of enzyme required for complete protection of 1 tg of X phage DNA for 1 hr from digestion with R-HpaI. Nucleotide sequencing Nucleotides were sequenced by the dideoxy chain termination method (28), except that 7-deaza-dGTP was used instead of dGTP. Restriction fragments were isolated from the HpaI restriction-modification genes and subcloned into the appropriate sites of the vectors Ml3mpl8 and mpl9. Alternatively, deletion derivatives were generated from the subclone containing the whole HpaI system using a kilo-sequence deletion kit. Overproduction of R HpaI Two E. coli recombinants, named E. coli RR1-HPA1-1 and HB1O1-HPA2, were cultured in 20 ml of LB broth (23) at 30°C until the absorbance at 600 nm was 1.0-2.0, heated at 42°C

for 15 min, then cultured at 37°C for 5 hr. The cells were harvested, suspended in 4 volumes of buffer A [20 mM potassium phosphate (pH 7.5) containing 10 mM 2-mercaptoethanol, 5% glycerol, and 0.15% Triton X-100], sonicated for 10 min, and centrifuged at 100,000xg for 30 min. The supernatant was assayed for R HpaI activity.

Purification of R HpaI overproduced by E. coli HB101-HPA2 First, 1.3 g of E. coli HB11-HPA2 cells was suspended in 3 ml of buffer A. The suspension was sonicated for 10 min (200 W, 1.1 A) and then centrifuged at 100,000xg for 30 min. To the supernatant, 1.4 g of solid ammonium sulfate was added. The resulting precipitate was dissolved in buffer B [20 mM potassium phosphate (pH 7.5), 10 mM 2-mercaptoethanol, and 5 % glycerol] and dialyzed against the same buffer. The R * HpaI activity was purified by chromatography on a column of phosphocellulose (Whatman P-1) and then a column of Sephadex G-75 (Pharmacia). The active fractions were concentrated to 1 ml with Centricon 10 (Amicon Corp.), mixed with 1 ml of glycerol, then stored at -20°C until use.

SDS-PAGE SDS-PAGE was performed by the method of Laemmli (29) with a 15 % polyacrylamide slab gel. Lysozyme (Mr 14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (42,700), bovine serum albumin (66,200), and phosphorylase b (97,400) were used as molecular weight markers (Bio-Rad Lab.).

RESULTS AND DISCUSSION Analysis of the N-terminal sequence of R HpaI Twenty-two amino acids, Met-Lys-Tyr-Glu-Glu-Ile-Asn-PheLys- Val-Pro-Val-Glu-Ser-Pro-Tyr-Tyr-Pro-Asn-Tyr-Ser-Gln, were identified in the N-terminal sequence of purified R HpaI.

(A)

I

ER ET

Bs H3 H3

Bs ET

pHPAI

SC

Ba

Activity

Kp

ER

0 to in R M R M

in vio

a.

(B)

+

+

+ +

__-

+-_-

0

Fig.

1. Southern

hybridization

of H.

parainfluenzae

chromosomal DNA. DNA

was digested with EcoRI, BamHI, HindIII, NruI, and KpnI, transferred from a 0.7% agarose gel to a nylon membrane, and hybridized with a synthetic probe labeled with 32p. EcoTI4I-digested lambda DNA was used as the size marker.

1

2

3

44

(kb)

Fig. 2. Restriction map of plasmid pHPA1. (A), Plasmid pHPAl was digested with various restriction endonucleases. The open box and the line represent the 3.8-kb NruI fragment and the pBRH vector, respectively. ER, EcoRI; ET, EcoT14I; Bs, BspHI; H3, Hindm; Ba, Ball; Sc, ScaI; Kp, KpnI. (B), The enzyme activities of various fragments were assayed in vivo and in vitro as described in 'MATERIALS AND METHODS'.

Nucleic Acids Research, Vol. 20, No. 4 707 A DNA probe consisting of 37 bases, 3'-TACTTTATGCTTCTTTAITTGAAGTTTCAIGGICAIC-5' ('I' indicates inosine residue), was synthesized based on this N-terminal amino acid sequence.

Isolation of the HpaI restriction-modification genes Southern hybridization of H. parainfluenzae chromosomal DNA with the synthetic DNA probe resulted in NruI digestion fragments of 3.8 kb (Fig. 1, arrow). The fragments were extracted from the agarose gel, and introduced into the PvuH

0.5

o

R.HpaI

1.0

1.5

co

Hpa

2.0

site of a new vector, plasmid pBRH. This vector, which two HpaI linkers [d(G-T-T-A-A-C)] were inserted into both the EcoRV and NruI sites of the pBR322, was constructed to isolate the M * HpaI gene. The recombinant plasmids were prepared from many transformants, and a plasmid, pHPA1 (Fig. 2), that was not digested by an excess of R HpaI, was selected. The clone carrying the pHPA1 was not infected by virulent X phage (titre, about 1 x 10-6 to 1 X 10-5). The restriction map of the pHPA1 (Fig. 2A) and the in vivo enzyme activities of various fragments (Fig. 2B) showed that the HpaI restriction-modification genes were in a 2.5-kb EcoRI fragment of the pHPA1. The in vitro enzyme activities, however, were not detected. The genes encoding these enzymes are transcribed with a native promoter

M S

2.5 (kb)

...

R.Hpal

M.Hpal

..

(kDa) 97- _.ow

Fig. 3. Organization of the HpaI system. The restriction map of the 2492 bpEcoRI fragment and the orientiation of the HpaI restriction-modification genes are shown.

66

-

43-

(Pv/Sr)

31-

22

Hp.

, ....

-

14-

Fig. 5. Estimation of the molecular weight of the purified R HpaI from E. coli HB101-pHPA2 cells by SDS-PAGE. Lane M, molecular weight markers (cft MATERIALS AND METHODS); lane S, R-HpaI purified from E. coli HB101-pHPA2. 0.9-kb BapHI blunt-ended f rag.

LpPL-lambda digested

2.5-kb UcoRI f rag.

LpKNH1

vector

with Ipal

vector digested

A

with UcoRI

Block I

M-Hpal M-HincII M-Accl M-TaqI

M-PsCI M-PaeR7 M.CviBIII

pHPA2 (7.3 kb) I

(HP/so)

x R

(position)

VLDPFVGSGTLNFV C ILEPSSGNGVFLDS L VLEPAFGLGVFSRAIL VLEPACAHGPFLRE A ILDAGAGVGSLTAA F LLEPSFGGGDFLLP 1 ILEPSCGTGEIISE C

(i96-211) (34-48)

(55-70) (43-60) (61-75)

(22-36) (53-67)

Block II

(position)

(23-32) (86-95) (118-127) (100-109) NKAILNPPYL (147-156) DFVVGNPPYV (115-124) DFIVGNPPYV (114-123)

DLIITDPPYN DSIIGNPPYV DGIICNPPYF DLILGNPPYG

Block III

LKPGGVLVFVVPATW (154-168) LKSGGELVAITPRSF (189-204) LSAGGNLGFICADRW (161-176) LKEDGILAFIIPSTI (155-169)

LPL

ZHR

B M BamH I

HLNRSGIGIDTNKEYIEMA KPH-TQKPEALYERMILASSNEGDIPLDPFVGSGD L DIVFDPFMGSGTTAKMAALNNRKYIOTEISK3YCDIA FQHPAIFPZKLAZDHILSWSBND

M-Cfr9

GABFATFPTELIRPCILAITKPGDYVLDPFFGIOTVGVVCQQEDRQYVGIELNP3YVDIA

M*HpaI

(HP/Be)

M-SmaI

GSHFAVFPRAMARLCVLAG8RPGGKVLDPVFG 1TTGWVCL DRECV0IELNE3YASLA

MRsrI

VG8PTYQPAAVIXRLVRALOHPGSTVLDFFAGSGVTARVAIQEGRNSICTDAAPVFKZYY

M.EcaI

TGYPTEKNFNMMKLIVGASSiIPODLVIDPFCGSGSTLHAASLL2RKwIOIGDESLFAAKTV

P oAh I NPARFPAKLP3FFI4RMLTEPDDLVVDIFGGSNTTGLVAERESRKWI SFEMKPZYVAAS Pw

(A)

(position)

LKPHGTIYIFMGMKY (64-78) LKVGGELIFICPDYF (167-181) LSQNGRCAYIIPSEF (136-150)

(171-230) ( 179 - 2 38) (224-283)

(225-284) (221-280) (320- 379) (244 - 303)

(B)

Fig. 4. Construction and restriction maps of plasmids pHPAl-l and pHPA2. Plasmid pHPAl-l (A) carries the R HpaI gene from plasmid pHPAl and the PL promoter and ampicillin-resistance gene from pPL-lambda vector. Plasmid pHPA2 (B) contains the genes encoding the HpaI system from pHPAl and the PL promoter and ampicillin-resistance gene from the pKHl vector. ER, EcoRI; Bs, BspHI; Pv, PvuI; Nr, NruI; Hp, HpaI; PL, PL promoter region; Ap, ampicillin-resistance gene; M, M-HpaI gene; R, R HpaI gene.

Fig. 6. Comparisons of the deduced amino acid sequence of M -HpaI with published sequences of other methyltransferases. (A), Comparison with other adenine methyltransferases. Amino acid sequences of homologous blocks are shown, together with the numerical positions of each block. (B), Comparison of regions of M * HpaI highly homologous with regions of other methyltransferases. The boxed sequence in M * HpaI indicates the block I in Fig. 6A. The bold and underlined letters indicate the identical and functionally similar amino acids to those in M HpaI, respectively.

708 Nucleic Acids Research, Vol. 20, No. 4 from H. parainfluenzae, so these are probably not expressed in E. coli enough for the detection in our assay conditions.

Nucleotide sequence of the HpaI restriction-modification genes The nucleotides, consisted of the 2492 bp-EcoRI fragment containing the HpaI restriction-modification genes, were sequenced. Figure 3 shows the organization of the HpaI system. Two open reading frames (ORFs) were found oriented in the same direction. The R * HpaI gene was 765 bp long (coding for positions 368 to 1132), corresponding to a protein of 254 amino acids. The N-terminal amino acid sequence deduced from the R * HpaI gene and that of the purified R * HpaI were completely identical. The M HpaI gene was 945 bp long (positions 1117 to 2061), corresponding to a protein of 314 amino acids. The molecular weight of this protein predicted from the M * HpaI gene was identical to that previously reported (11). These genes overlapped by 16 bp. Shine-Dalgarno-like sequences were found in both genes (positions 358 to 361, a sequence 5'-AGGA-3', for the R HpaI gene and positions 1100 to 1103, 5'-GAGG-3', for the M * HpaI gene). Putative TATA box-like sequences were detected upstream of both genes (positions 328 to 333, 1081 to 1084, and 1094 to 1098). The GC content of both genes was below 30%. The gene organization of the HpaI system has been reported by Wilson (30). The number of amino acids that we deduced from the nucleotide sequences agreed with his results. However, the nucleotide sequences of the genes encoding the HpaI system were not reported there. Some of the nucleotide sequence of the ORF that was upstream from the R HpaI gene was studied using the Gene/Protein database. The deduced amino acid sequence of the ORF had 70% homology with the sequence of the E. coli 5-methyltetrahydrofolate homocysteine methyltransferase (31). It is not clear whether these genes form an operon. However, it is of interest that this homocysteine methyltransferase gene is near the HpaI restriction-modification genes.

Cloning of the R HpaI gene into E. coli cells carrying the M-HincH gene M HincH also methylated the recognition site of the HpaI restriction-modification system. Therefore, the R HpaI gene should be expressed in E. coli cells carrying the M Hincd gene. The plasmid pHPAl was digested with BspHI and treated with T4 DNA polymerase. The 0.9-kb BspHI blunt-ended fragment was introduced into the HpaI site of pPL-lambda vector, resulting in a plasmid named pHPA1-1 (Fig. 4A). This plasmid contained the PL promoter from X phage DNA, so plasmid pNT203 (21) was used to control expression of the gene ligated downstream of the promoter. Plasmid pAH-M.Hc (13), which inserted the M HincII gene ligated downstream of the lac promoter into the EcoRV site of the pACYC184 vector, has been reported (13). The three plasmids pHPAI-1, pAH-M.Hc, and pNT203 were cloned in the same E. coli RR1 cells, and the new strain was named E. coli RR1-HPA1 1. R * HpaI protein was expressed and induced in E. coli RRl-HPA1 l cells as described under 'MATERIALS AND METHODS'. The total activity in vitro was 400,000 U/g cells. This activity was 10-fold that from H. parainfluenzae cells. The R HpaI gene was cloned in E. coli RR1 cells carrying the M * HincII gene. However, the overproduced R * HpaI levels were small and the band corresponding to the overproduced R * HpaI was not detected by -

-

SDS-PAGE.

Overproduction and purification of R HpaI from E. coli RR1-HPA2 The 2.5-kb EcoRI fragment containing the genes encoding the HpaI system (cf. Figs. 2 and 3) was introduced into the EcoRI site of the pKHl vector, resulting in plasmid pHPA2 (Fig. 4B). The pKHl vector has been previously reported (20). The two plasmids pHPA2 and pNT203 were cloned in E. coli HBIOI cells and the strain was named E. coli HBO10-HPA2. The activity heatinduced R * HpaI from this strain in vitro was 4,000,000 U/g cells. This activity was 100-fold that from H. parainfluenzae cells and 10-fold that from E. coli RR1-HPA1 1 cells. R HpaI protein was purified from the supernatant by chromatography through Pcellulose and gel filtration through Sephadex G-75 as described in 'MATERIALS AND METHODS'. The enzyme preparation migrated mostly as single band during SDS-PAGE (Fig. 5). The molecular weight of the purified R -HpaI was 29,500, which agreed well with that of the amino acid sequence deduced from those of the DNA sequence (Fig. 3). Comparisons of amino acid sequences The deduced amino acid sequence of M *HpaI was compared with the published sequences of other methyltransferases (2, 8, 13, 32-40) using the Gene/Protein database (Fig. 6). The deduced sequence of the M * HpaI gene was found to contain a segment of tetra-amino acids, Asp-Pro-Pro-Tyr, that has been identified as a characteristic of N6-adenine methyltransferases (the block II in Fig. 6A). We previously reported (13) that there are three homologous regions, blocks I, II, and Im, among the TNNA-specific adenine methyltransferases (Fig. 6A). M HpaI also had these homologous regions. However, the order of each block differed from that of other adenine methyltransferases (Fig. 6A). In the TNNA-specific adenine methyltransferases other than M * HpaI, these regions find towards the N-terminus. However, each block in the M - HpaI gene was distributed throughout the sequence. Investigations into the functions of these blocks are now in progress. M HpaI has also homology with the six methyltransferases with published sequences (8, 36-40, Fig. 6B). M-HpaI has homology with the N4-cytosine methyltransferases rather than with the N6-adenine methyltransferases in the regions containing block I. The SNN(N)NNS sequence is common in the recognition of these methyltransferases. However, these regions are probable the binding sites of DNA or S-adenosylmethionine, because these are found in both adenine and cytosine methyltransferases (41,42).

ACKNOWLEDGMENTS We thank Mr. M.Ito of Bioproducts Development Center, Takara Shuzo Co., Ltd., for the N-terminal amino acid sequence analysis.

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