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Bci528I, a new isoschizomer of EcoRI isolated from Bacillus circulans 528

Sung-Ryong Ra 1 & Myong-Suk Kim 1 & Chon-IL Paek 1 & Yong-Chol Pak 1 & Song-Hui Pak 1 & Hyong-Bom Pak 1 &
Kum-Chol Ri 2

Received: 28 September 2018 /Accepted: 28 February 2019
# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2019

Abstract
Bacillus circulans 528 produces a restriction endonuclease, Bci528I which is an isoschizomer of EcoRI. We purified the enzyme, using Sephadex G-150, Phospho-cellulose, DEAE-cellulose, Hepharin-Sepharose CL-6B chromatography. The specific activity of Bci528I was 29,400 U/mg·protein. Bci528I recognizes 5′-GAATTC-3′ in dsDNA and cleaves between G and A of the recognition sequence, producing a symmetric four base 5′overhang.

Introduction

Since the discovery of the first restriction enzyme in Escherichia coli, over 4000 type II restriction endonucleases have been discovered in different bacterial species and virus, and more than 250 different specificities have been character- ized (Meselson and Yuan 1968; Swaminathan et al. 1996; Roberts et al. 2015).
Restriction endonucleases are enzymes that have ability to cleave double-stranded DNA. Restriction endonucleases are parts of restriction-modification (RM) systems which occur ubiquitously among prokaryotic organisms. RM systems play important roles in protecting bacterial cells against bacterio- phage attack, because the foreign DNA is highly specifically cleaved by the restriction endonuclease if it contains the rec- ognition sequence of the endonuclease.
Restriction endonuclease can be classified into type I, II, III, or IV, depending on the subunit composition, the cofactor requirement and the mechanism of action (Roberts et al. 2015). Typically, type II restriction endonuclease (type IIP group) recognizes palindromic sequences, ranging from 4 to 8 bp and cleaves the double strands of DNA within or imme- diately adjacent to the recognition sites (Roberts et al. 2003).
Typical representatives of the group are EcoRI, EcoRV, and BglI. However, some type II restriction endonucleases have unusual properties of recognition and cleavage. Type IIS re- striction endonucleases recognize asymmetric sequences and cleave the DNA outside of the recognition sequences, for ex- ample FokI (Szybalski et al. 1991). And then, type IIB endo- nuclease (BcgI, BaeI) cleave on both sides of recognition sites, producing short DNA fragments (Sears et al. 1996).
Type II restriction endonucleases are both indispensable tools for manipulating DNA and excellent enzyme models for studying sequence specific interaction with DNA. Because of their importance, type II restriction endonucleases have been studied in great detail.
However, to the best of our knowledge, there has never been any report on identification of isoschizomer of EcoRI in the genus Bacillus.
In this study, we report the isolation and characterization of Bci528I, a new member of the restriction endonuclease type IIP group, which recognizes the palindromic sequence 5´- GAATTC-3′ and cleaves between G and A of the recognition sequence. The recognition sequence and cleavage site of Bci528I are the same as those of EcoRI. Thus, Bci528I is the isoschizomer of EcoRI.

* Sung-Ryong Ra [email protected]

Materials and methods

1

Research Institute of Biotechnology, Faculty of Life Science, Kim Il Sung University, Pyongyang, Democratic People’s Republic of Korea

Bacterial strain, enzyme, and plasmids

The bacterial strain B. circulans 528 (No 528) was isolated

2 Korean Center for Culture Collection, Pyongyang, Democratic People’s Republic of Korea
from soil and identified using the same procedure that was detailed in Bergy’s Manual of Systemic Bacteriology, based

on its morphological, physiological, and biochemical charac- teristics (Vos et al. 2009). The strain has been deposited as KCCC11124 in WFCC joined Korean Center for Culture Collection. The cells were grown in 500-ml flasks with 50 ml of nutrient medium (5 g Bacto yeast extract, 10 g Bacto tryptone, 5 g glucose per L) at 30 °C with vigorous aeration to late log phage (5 × 108~109 cells/ml). The enzyme and plasmids used in this study were purchased from Sigma.

Enzyme purification

The frozen cells were disrupted by sonication for 20 × 30 s bursts at 100 W under careful cooling and the debris was removed by centrifugation (12,000 rpm, 90 min). Nucleic acids were removed from the crude extract by gel filtration chromatography on Sephadex G-150 (pharmacia) at high ion- ic strength (1 M NaCl). The active fractions were adjusted to 70% saturation with solid ammonium sulfate, stirred for 30 min, and the precipitate was then collected by centrifuga- tion. The pellet was suspended in 10 ml buffer and dialyzed. The endonuclease Bci528I was purified from dialyzed sample by the following chromatographic steps: Phospho-cellulose (WhatmanP-11), DEAE-cellulose (Whatman DE-52), and Heparin-Sepharose CL-6B (Pharmacia) column chromatogra- phy (Greene et al. 1978).
To determine the purity of Bci528I, samples in different purification steps and protein size marker kit (pharmacia) were subjected to SDS-PAGE analysis (Laemmli 1970).
One unit of the enzyme activity is defined as the amount of enzyme required to totally digest 1 μg of λ DNA under the standard reaction conditions in 1 h at 37 °C.
The restriction enzyme isolated from B. circulans 528 was named Bci528I according to the nomenclature of restriction enzyme (Roberts et al. 2003).

Enzyme characterization

The effect of reaction mixtures on enzyme activity was inves- tigated with a set of buffers for restriction enzymes, namely R, B, O, G, and W.
Reaction buffers:

R: 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 50 mM NaCl, 7 mM 2-mercaptoethanol
B: 10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 7 mM 2- mercaptoethanol
G: 10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 50 mM NaCl, 7 mM 2-mercaptoethanol
O: 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 100 mM NaCl, 7 mM 2-mercaptoethanol
W: 10 mM Tris-HCl, pH 8.5, 10 mM MgCl2, 100 mM NaCl, 7 mM 2-mercaptoethanol

Determination of the Bci528I cleavage site

The cleavage site of Bci528I was determined with the fluores- cently labeled dideoxy chain-termination method in ABI PRISM 310 Genetic Analyzer, the capillary sequencer. The plasmid pGEM-3Zf (+) DNA was isolated according to the procedure used by Sambrook and Russell (2001) and served as template in a polymerization reaction using Ampli Taq FS DNA polymerase. The end-labeled reaction kit with dichlororhodamines was used to provide a tracer for the prod- ucts of the polymerization process. A POP-6 gel was subject- ed to electrophoresis.
The primers used in DNA analysis were as follows:

M13 directed sequence: 5′-TGTAAAACGACGAC GGCCAGT-3′
M13 reverse sequence: 5′-CAGGAAACAGCTAT GACC-3′

Results

The identification of bacterial strain producing Bci528I

Fifty bacterial strains from soil samples were isolated and restriction activities were detected in cell-free extracts of iso- lated B. circulans strains (Fig. 1). Figure 1 indicates that ten- fold diluted crude extract of No. 528 has a restriction activity in reaction buffer of middle concentration of salt (10 mM Tris- HCl, pH 7.6, 10 mM MgCl2, 50 mM NaCl, 7 mM 2- mercaptoethanol).
The strain No. 528 was identified as Bacillus circulans, based on its morphological, physiological, and biochemical characteristics (Vos et al. 2009). Restriction endonuclease iso- lated from B. circulans was designated Bci528I.

Enzyme purification

To purify restriction endonuclease Bci528I, we carried out such chromatographic steps as Sephadex G-150, Phospho- cellulose P-11, DEAE-cellulose DE-52, and Heparin- Sepharose CL-6B column chromatography. Figure 2 shows the elution profile of Heparin-Sepharose CL-6B column chro- matography and Fig. 3 shows SDS-PAGE of molecular weight markers and samples after all purification steps of Bci528I. The final fractions did not contain non-specific nu- cleases. In SDS-polyacrylamide gel, Bci528I migrates as sin- gle band, indicating that the Heparin-Sepharose CL-6B frac- tion is highly purified and virtually pure. A summary of the purification is presented in Table 1. From 5 g of wet cell paste, we were able to obtain 18,500 U of homogenous enzyme

Fig. 1 The activities of restriction endonucleases in crude extracts of several strains. 1, λ DNA; 2, λ DNA + No.505 (crude extract 1 μL); 3, λ DNA + No.505 (crude extract 3 μL); 4, λ DNA + No.528 (crude extract 1 μL); 5, λ DNA + No.528 (crude extract 3 μL); 6, λ DNA + No.535 (crude extract 1 μL); and 7, λ DNA + No.535 (crude extract 3 μL)

Fig. 3 SDS-PAGE of enzyme fractions obtained during purification procedure. 1, crude-extract; 2, Sephadex G-150 fraction; 3, Phospho- cellulose fraction; 4, DEAE-cellulose fraction; 5, Heparin-Sepharose CL-6B; M, molecular markers

preparation with an overall yield of 21.5%. The specific ac-

tivity of restriction endonuclease Bci528I was 29,400 U/mg·
protein (Table 1).

Enzyme characterization

We investigated the influence of reaction buffer composition on the enzyme activity of Bci528I. As shown in Fig. 4, λ DNA was totally digested in G and R buffer, but partially digested in B, O, and W buffer, respectively. Restriction activity of Bci528I was strongly inhibited due to the excessive amounts of Tris-HCl and NaCl in O buffer, and also inhibited by no presence of NaCl in B buffer. The results show that the opti- mal buffer composition suitable for Bci528I restriction
activity was 10 mM Tris-HCl (pH 7.6~8.0), 10 mM MgCl2, 50 mM NaCl, and 7 mM 2-mercaptoethanol.

Determination of recognition sequence and cleavage site of Bci528I

In order to determine the recognition sequence of Bci528I, the electrophoretic figure of λ DNA digested by restriction endo- nuclease Bci528I was compared with that described in the instruction book for restriction endonuclease. The result indi- cates that Bci528I digestion pattern on λ DNA is identical to EcoRI pattern. A double digestion with Bci528I and EcoRI on bacteriophage λ DNA confirmed that Bci528I is an isoschizomer of EcoRI recognizing nucleotide sequence 5′- GAATTC-3′ (Fig. 5).
The cleavage site of Bci528I was determined using the primer extension method with M13 primers and plasmid pGEM-3Zf (+) DNA as template. As shown in Fig. 6, the terminal nucleotides of these two chains are the same as 5′- NNNNNGAATTA-3′. In these terminal sequences, 3′-termi- nal A is the base that was non-specifically added by Taq DNA polymerase.
Based on all information regarding to the recognition se- quence and cleavage site of restriction endonuclease Bci528I, it turned out that Bci528I recognizes the sequence 5′-GAAT TC-3′ and cleaves between G and A of the recognition se- quence, producing a symmetric four-base 5′overhangs.
The results demonstrate that Bci528I is a type IIP restric-

Fig. 2 Chromatogram of Bci528I on Heparin-Sepharose CL-6B column. a A280. b Enzyme activity. c KCl concentration
tion endonuclease using the same nomenclature as described previously (Roberts et al. 2003).

Table 1 Purification of the restriction endonuclease Bci528I from B. circulans (5 g wet weight)
-3
Purification steps Total protein (mg) Total activity (U × 10 ) Specific activity (U/mg protein) Degree of purification (folds) Yield (%)

Crude extract 687 86.1 125 1 100
Sephadex G-150 33.8 67.8 2010 16.0 78.7
Phospho-cellulose 6.62 46.3 6990 55.8 53.8
DEAE-cellulose 2.11 28.7 13,600 108 33.3

Heparin-Sepharose- CL-6B
0.63
18.5
29,400
235
21.5

The Bci528I enzyme has been registered on the official REBASE website (http://rebase.neb.com) under enzyme number 18749.

Discussion

The present study was designed to isolate restriction en- donuclease Bci528I from B. circulans 528 and to deter- mine the recognition sequence and cleavage site of that enzyme.
The restriction activity in crude extract of B. circulans 528 identified from soil was discovered. 18,500 U of Bci528I with a specific activity of 29,400 U/mg protein was obtained from 5 g of wet cell paste.

Fig. 4 Activities of Bci528I in different buffers. 1, B; 2, G; 3, O; 4, R; 5, W

From a practical point of view, Bci528I restriction endonuclease is quite useful. One hundred millimolar of Tris-HCl was required for complete restriction activ- ity of EcoRI, whereas 10 mM Tris-HCl was optimal to Bci528I (Greene et al. 1978). The results of this study indicate that the endonuclease Bci528I will be widely used as a tool for gene cloning and DNA sequence analysis because Bci528I was required lower concentra- tion of Tris-HCl for cleavage of dsDNA, in comparison with EcoRI.
The most interesting finding is that Bci528I, an isoschizomer of EcoRI from E. coli, recognizes 5′-GAAT TC-3′ in dsDNA and cleaves between G and A of the recog- nition sequence. It is of interest that two microorganisms be- longing to distinct genera produce a restriction endonuclease having the same specificity.

Fig. 5 Electrophoretic profiles of digestion of λ DNA by Bci528I and EcoRI. 1, λ DNA; 2, λ DNA/Bci528I; 3, λ DNA/EcoRI; and 4, λ DNA/
Bci528I + EcoRI

Fig. 6 Sequence analysis of the terminal sequence of pGEM-3Zf (+) DNA cleaved with Bci528I

It was well known that restriction endonucleases recogniz- ing long nucleotide sequences evolved from a restriction en- zyme recognizing short nucleotide sequences (Mannarelli et al. 1985). However, no restriction endonucleases recogniz- ing tetranucleotide were found in the genus Escherichia (Janulaitis et al. 1988; Roberts et al. 2010). ecoRI gene and the genome of E. coli have the A+Tcontents of 65% and 49%, respectively (Greene et al. 1981; Newman et al. 1981). Thus, it may be supposed that ecoRI gene was horizontally trans- ferred from distinct bacterial species to E. coli. On the other hand, the bacteria in the genus Bacillus have restriction endo- nucleases that recognize different lengths of nucleotide se- quence. After discovering the Bci528I, an isoschizomer of EcoRI in the genus Bacillus, we may hypothesize that the ecoRI gene is the product of horizontal gene transfer from the genus Bacillus to the genus Escherichia followed by the appearance of restriction enzymes which recognize the hexanucleotide sequence 5′-GAATTC-3′ in the genus Bacillus.
More recent attention has been focused on the investigation of horizontal gene transfer in evolution of bacterial genome (Verhikas and Medini 2010; Kita et al. 2003). Therefore, fur- ther studies on the current topic need to be undertaken to understand the relationship between ecoRI and bci528I.
Acknowledgements We thank Dr. Chol-Min Pak of the research institute of biotechnology, academy of science, the DPR of Korea for determina- tion of recognition sequence and cleavage site.

Authors’ contribution Sung-Ryong Ra conceived and designed the ex- periments. Myong-Suk Kim and Chon-Il Paek conducted experiments with support from Hyong-Bom Pak, Kum-Chol Ri, and Yong-Chol Pak. Song Hui Pak analyzed the data. Chon-Il Paek wrote the manuscript. All authors read and approved the final manuscript.

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