Fused eco29kIR- and Mgenes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems
© Mokrishcheva et al; licensee BioMed Central Ltd. 2011
Received: 3 August 2009
Accepted: 3 February 2011
Published: 3 February 2011
The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.
Fusion of eco29kIR and M ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. In vitro the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. In vivo new RMS can provide a 102-fold less protection of host cells against phage invasion.
We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.
DNA restriction-modification systems (RMS) are prokaryotic tools against invasion of foreign DNAs into cells . They play an important evolutionary role as subcellular barriers restricting horizontal gene transfer and thereby providing microbial biodiversity. Usually, RMS comprise of a restriction endonuclease (REase) and modification DNA methyltransferase (MTase) enzyme recognizing the same short 4-8 nucleotide sequence. RMS functioning includes methylation of recognition DNA sequences by MTase. All non-modified sites can be cut by a cognate REase . Type II REases are indispensable tools in creating recombinant DNA molecules . Their widespread practical application has stimulated research to discover and characterize more of these systems. Currently, more than 6000 different sequences corresponding to REases of type II alone are listed in REBASE, the database holding all known and many putative RMS .
The high number of known RMS is reflected also in high diversity of their organization or functioning and, hypothetically, in multiplicity of their evolutionary pathways. One of these pathways could be fusion of preexisting ORFs with formation of a gene capable of producing a protein with an array of new activities and functions. It could be suggested that type IIC RMS carrying both REase and MTase in a single polypeptide might appear by this mechanism . Here we report direct evidence how a fully functional type IIC REase could appear by fusion of the appropriate genes as a result of a few point mutations.
Apparently, a similar mechanism of gene fusion via natural point mutations/deletions/insertions/invertions/translocations could give type IIC REases as well as bifunctional MTases like SsoII-related enzymes and bifunctional MTases like FokI and LlaI [4, 11, 12]. Apart from methyltransferase, these proteins also possess separate domains involved in transcriptional regulation (SsoII) or methylation (FokI and LlaI). It should be noted that gene fusion is facilitated by the head-to-tail gene orientation, which is quite common for many RMS. For example, the following type II RM systems are organized in this way: AccI, BanI, Bsp6I, BsuBI, Cfr9I, DdeI, EagI, EcoPI, EcoP15, EcoRI, FnuDI, HaeIII, HgiBI, HgiCI, HgiCII, HgiDI, HgiEI, HgiGI, HhaII, HincII, HindIII, HinfI, HpaI, MboII, MwoI, NcoI, NdeI, NgoMI, NgoPII, NlaIII, PaeR7I, RsrI, SalI, Sau3A, Sau96I, TaqI, TthHB8I, XbaI, and XmaI, and many more can be found in RMS database [3, 12]. Thus, their unidentified type IIC bifunctional derivatives could exist in nature.
Construction of overproducing strain and purification of RM.Eco29kI
To construct an overproducing strain for the protein RM.Eco29kI, ORFs of Eco29kI REase and MTase were amplified from the natural plasmid pECO29 where they are oriented as shown in Figure 1A,. On this plasmid the Stop codon of Eco29kI REase and the Start codon of Eco29kI MTase overlap. Subsequently they were cloned in the same orientation into the pET19mod vector. By site-directed mutagenesis their separating Stop and Start codons were substituted for 2 Glycine codons, as shown in Figure 1B, thus forming the eco29kI.RM gene. The resulting RM.Eco29kI polypeptide contains both REase and MTase enzymes joint by a flexible 2 Glycine hinge and 6 His-tag on its N terminus.
The purification scheme for RM.Eco29kI was based on affinity (Ni-CAM, nickel chelate affinity matrix, Sigma) chromatography. From a Ni-CAM column RM.Eco29kI was eluted by linear steps of 20, 50, 75, 100 and 150 mM imidazole. Finally, 200 ml of cell culture gave about 0.4 mg of >98% purified enzyme (Figure 1C) with a molecular weight of ~67 kDa.
When the Stop codon of Eco29kI REase gene was substituted for Glycine without modifying the first Methionine codon of Eco29kI MTase gene, expression of M.Eco29kI, but not RM.Eco29kI, was detected (unpublished observations). Hypothetically, it might happen due to a pronounced secondary structure of mRNA in the region corresponding to R.Eco29kI ORF or, as it was reported personally by M. Nagornykh, to transcription initiation from an alternative promoter closely preceding M.Eco29kI ORF .
Characterization of RM.Eco29kI REase activity
Comparison of reaction optima for R.Eco29kI and fused RM.Eco29kI.
Characterization of RM.Eco29kI MTase activity
The biochemical characterization of Eco29kI MTase is presented in . The enzyme methylates the second Cytosine in the sequence CC Me GCGG. Specificity of RM.Eco29kI MTase activity was proved to be the same, because there was no incorporation of labeled methyl groups into substrates pretreated with M.Eco29kI enzyme and non-labeled AdoMet. The optima of reaction conditions also remained unchanged: both enzymes showed their maximal activities at 50 mM NaCl; 5 mM EDTA; pH 7.0-8.5, and 37°C. Under optimal conditions M.Eco29kI had a specific activity of 10 AU/pMol, whereas RM.Eco29kI had 8 AU/pMol amounting to 80% of the native value. Thus, after fusion with R.Eco29kI, activity of its MTase part was almost unchanged.
Characterization of RM.Eco29kI functioning in vivo
Characterization of RM.Eco29kI behavior in vitro
Nomenclature of the RM.Eco29kI enzyme
Major properties of proved type IIC enzymes.
Type of MTase
Presence of separate M gene
B, G, S
B, G, H, S
(10/12) CGANNNNNNTGC (12/10)
yes, two genes
B, G, S
(8/14) CCANNNNNNGT (15/9)
E, G, S
B, G, P
(7/13) GAYNNNNNRTC (14/9)
TCCRAC (20/18) or TCCRAC (21/19)
B, G, S
(7/12) GAACNNNNNCTC (13/8)
B, G, S
(8/13) CACNNNNNNTCC (12/7)
The role of gene fusion in molecular evolution of RMS
In our study a fully functional hybrid polypeptide was generated by fusion of Eco29kI REase and MTase proteins. New protein had REase and MTase specificities of the original enzymes. Its MTase activity was almost unchanged and amounted to 80% of that of the M.Eco29kI under optimal reaction conditions for both enzymes: 50 mM NaCl; 5 mM EDTA; pH 8.0, and 37°C . Its REase activity was decreased by three times, which could be attributed to increased steric hindrance in interaction with substrate. Besides, the reaction optimum for REase activity of RM.Eco29kI differed from that of the R.Eco29kI as follows: 100 mM less for [NaCl], 50 mM less for [KCl], and 1 unit less for pH (Table 1). The particular reason for this shift is unclear because many physical and chemical properties of the protein, such as molecular weight, isoelectric point, total charge, geometry of the protein, surface charge distribution, etc., were different from those of the original proteins. Hypothetically, the gap between their properties could yield as a result of the natural selection, after many generations, a novel protein with altered biochemical properties and different functions in the cell.
It can be predicted that novel type IIC RMS may appear via fusion of genes from bipartite RMS having head-to-tail gene organization. This type of gene organization is quite common for RMS of all types. Type I RMS such as CfrA, EcoA, EcoB, EcoD, EcoE, EcoK, EcoR124, StySB, StySI, StySP, and StySQ; type II RMS such as AccI, BanI, Bsp6I, BsuBI, Cfr9I, DdeI, EagI, EcoPI, EcoP15, EcoRI, FnuDI, HaeIII, HgiBI, HgiCI, HgiCII, HgiDI, HgiEI, HgiGI, HhaII, HincII, HindIII, HinfI, HpaI, MboII, MwoI, NcoI, NdeI, NgoMI, NgoPII, NlaIII, PaeR7I, RsrI, SalI, Sau3A, Sau96I, TaqI, TthHB8I, XbaI, and XmaI, all have head-to-tail gene orientation, thus being good candidates for originating new type IIC enzymes . These possibilities could be facilitated by increased affinity of DNA MTases for hemimethylated substrates in comparison with non-methylated ones, unlike their REase counterparts. Therefore, if a fused RMS appeared from preexisting bipartite RMS and its MTase activity was lower of the original enzyme, its propagation would be facilitated by facts that all recognition sites in the host genome were methylated or, after replication, hemimethylated by the preexisting enzyme and that it had increased affinity to hemimethylated substrates. Otherwise, decreased MTase activity of a new fused RMS could lead to appearance of unprotected recognition sequences in host genome, which will be cut by REase, host cells will die and this RMS will not propagate in a bacterial population.
In principle, any other adjacent gene of appropriate orientation could be fused with the REase or MTase part of RMS. In this case the fact that RMS elimination is lethal for cells would defend the fused ORF from being lost . It occurs because long-lasting endonucleolitic activity results in multiple cuts on non-protected genomic DNA, thereby killing cells that lack RMS. Thus, joining RMS genes would promote maintenance of a fused ORF and its spreading in bacterial populations, provided their functioning is not disturbed.
Practical applications of gene fusion for generating REases with novel recognition sites
Restriction enzymes are robust tools for the recombinant DNA technology. Despite the fact that more than 200 enzymes with different recognition sites have been isolated from various bacterial strains, many specificities have not been discovered [23, 26]. To create enzymes with altered recognition specificities, methylation activity-based selection (MABS) and target recognition domain (TRD) reassortment [23, 26] approaches were proposed. Using these techniques, bifunctional enzymes of type IIC such as Eco57I, AloI, PpiI, and TstI were manipulated to yield a generation with novel specificities. Most importantly, both of their activities operated on the same target sequence, thus providing a possibility to use the DNA-modification activity of these enzymes for the selection of mutants with altered sequence specificity. To enlarge the list of enzymes available for these manipulations, a gene fusion approach, similar to one used in this work, could be applied. By this procedure, hundreds of bifunctional enzymes could be created, e.g., from type I and III head-to-tail oriented RMS, thus giving an opportunity of significant contribution to the existing recognition specificities.
Altogether, our work presents an example of molecular mechanism for appearance of type IIC restriction-modification and SsoII MTase-related enzymes as well as other multifunctional proteins. It demonstrates that gene fusion could play an important role in evolution of restriction-modification systems and be responsible for enzyme subclass interconversion. Based on the proposed approach, hundreds of novel type IIC enzymes could be generated from head-to-tail oriented type I, II and III restriction and modification genes. These new bifunctional polypeptides could be useful for creating enzymes with altered recognition specificities. Lastly, our work shows that protein fusion can change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence, which, after many generations, gives a novel protein with both altered biochemical properties and different functions in the cell.
All strains used were grown in LB liquid medium with high aeration. For selection ampicillin (100 μg/ml) and kanamycin (25 μg/ml) were added to LB agar plates .
All genetic engineering methods were performed as described . Selection of transformants with plasmids carrying Eco29kI RMS genes and screening of recombinant clones were performed as elsewhere [30, 31].
Chemicals, Bacterial Strains, Plasmids and Enzymes
E. coli BL21(DE3) (Novagen) and Z-85 strains were used for transformation experiments . Plasmids pET19mod, pECO29, and p29k11 were used for a number of experiments [5, 7, 33]. Restriction endonucleases, T4 DNA ligase, and thermostable DNA-polymerase from Th. aquaticus were purified in our laboratory. Pfu DNA polymerase was purchased from New England Biolabs (UK). Protein and DNA ladders were obtained from Fermentas (Lithuania).
Strain BL21(DE3) (Novagen), transformed with plasmid p29k11 (Kmr) bearing eco29kIM gene was used as a host for transformation of plasmid containing eco29kI RM fusion gene .
(3H-methyl)-labeled AdoMet (15Ci/mMol) was purchased from Amersham Biosciences; unlabeled AdoMet, potassium phosphate, sodium chloride, potassium chloride, bovine serum albumin were from Sigma. Acrylamide, methylenebisacrylamide, Coomassie Brilliant Blue R-250 and Low Melting Agarose (LMP) were from BioRad. All other chemical reagents used were of the highest grade purity.
Construction of Eco29kI REase and MTase fusion RM.Eco29kI protein overproducing strain and biomass production
Eco29kI REase and MTase genes were amplified from the natural plasmid pECO29 with primers TTTGTCGAC ATGCACAATAAGAAATTTGATA (forward; containing start ATG codon shown in bold) and CCTGGATCCCTTTTAATTGAAGTTAGAGCACAA (reverse; containing stop TTA codon shown in bold), carrying SalI and BamHI sites, respectively, for subsequent cloning . Appropriately digested PCR product was cloned into pET19mod vector. Site-directed mutagenesis has been performed to fuse Eco29kI REase and MTase ORFs, using the high fidelity Pfu DNA polymerase and oligonucleotide primers:
Fus1: AAGAGTAATTTTACAGGAGGGAGATCATTAGAG and
A novel plasmid containing staggered nicks was generated. Following the thermal cycling, the reaction mixture was treated with DpnI REase that digests hemimethylated parental DNA, leaving behind the newly amplified nicked DNA with the mutation of interest. The mutation and eco29kI.RM gene were confirmed by sequencing. The resulting construct was referred to as p29RM.
The plasmid p29RM was introduced into E. coli BL21(DE3) strain. Cells were grown in LB medium supplemented with ampicillin and kanamycin at 20°C. After reaching OD590 = 0.6, the recombinant protein RM.Eco29kI synthesis was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM followed by overnight incubation at 20°C. Cells were harvested by centrifugation, frozen and stored at -70°C. All subsequent steps were carried out at 4°C.
Construction of natural pECO29 plasmid carrying fused Eco29kI REase and MTase genes
To make Eco29kI REase and MTase gene fusion on natural pECO29 plasmid, its BclI-PvuII fragment was substituted for BclI-PvuII fragment of p29RM plasmid, containing the gene fusion mutations. Resulting plasmid was referred as pECO29RM.
RM.Eco29kI-producing E.coli cells pelleted from 200 ml of culture were lysed by lysozyme treatment (1 mg/ml, 30 min at 4°C) in 5 ml of ice-cold buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM β-mercaptoethanol, 10 mM Imidazole, and 5% glycerol (v/v)) with protease inhibitor 1 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication. Triton X-100 was added to the lysate to a final concentration of 1%, and the lysate was subjected to centrifugation at 14,000 rpm, 4°C for 40 min to eliminate cell debris. The resulting supernatant was applied to a Ni-CAM (Sigma) column equilibrated with buffer A with 1% Triton X-100. After washing the column with buffer B (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 5 mM β-mercaptoethanol, 15 mM Imidazole, 5% glycerol (v/v), and 1% Triton X-100), the protein was eluted using linear steps (20-150 mM) of imidazole in buffer A with 0.1% Triton X-100. Active fractions with specific REase activity containing homogenous RM.Eco29kI (as judged by SDS-PAGE) were pooled and dialyzed overnight against storage buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 7 mM β-mercaptoethanol, 0.1 mM EDTA, 0.1% Triton X-100, 50% glycerol (v/v)) and stored at -20°C. The purified protein was analyzed by SDS-PAGE according to the method of Laemmli using Coumassie Blue R-250 (BioRad, USA) conventional staining .
Characterization of phage restriction by RM.Eco29kI RMS
To characterize RM.Eco29kI RMS ability to protect host cells against phage invasion different dilutions of phage λvir (100, 10-2, 10-4, 10-6) were spotted on bacterial lawns of BL21(DE3)xp29k11 (negative control), BL21(DE3)xp29k11+p29RM (with 0.03 mM IPTG), BL21(DE3)xpECO29RM (eco29kIRM gene under natural promoter) and BL21(DE3)xpECO29 (positive control) cells. Then minimal concentrations of phage λvir, capable to infect corresponding strains, were compared.
To test RM.Eco29kI DNA methyltransferase activity, we used DE-filters assay according to .
Analysis of Protein Concentration
Protein concentration was determined from the absorption at 280 nm on a Shimadzu UV-1601 spectrophotometer (Japan). An extinction coefficient was calculated by ProtParam tool http://www.expasy.ch: 94 240 M-1 cm-1.
RM.Eco29kI REase- and MTase reaction optima
Investigation of AdoMet influence on REase activity of RM.Eco29kI
To study the AdoMet effect on REase activity of RM.Eco29kI, 0.5 μg of phage φ80vir DNA was treated with 2-fold dilutions of 0.1 μg RM.Eco29kI at REase (50 mM NaCl, 10 mM MgCl2; 20 mM Tris-HCl, pH 7.5) and MTase (50 mM NaCl; 5 mM β-mercaptoethanol; 20 mM Tris-HCl, pH 8.0, with 10 mM MgCl2) optimal conditions with or without excess AdoMet (10 μM) for 1 h at 37°C. Reaction products were analyzed electrophoretically in 0.8% agarose gel.
The amount of enzyme required to transfer 1 pmol of (3H)-methyl groups to DNA per minute with saturating concentrations of substrates at 37°C has been taken as 1 AU of the RM.Eco29kI DNA methyltransferase activity. The amount of enzyme required to cut 1 μg of phage φ80vir DNA during 1 h at 37°C has been taken as 1 AU of the RM.Eco29kI restriction endonuclease activity. Both reactions were carried out under predefined optimal reaction conditions.
List of abbreviations used
open reading frame
target recognition domain
fused REase and MTase Eco29kI in one polypeptide
6 Histidine residues
- LB medium:
rotations per minute
polyacrylamide gel electrophoresis
The authors thank Prof. E.S. Gromova and N.A. Cherepanova for help in MTase activity characterization experiments and Dr. M.V. Zakharova for the kindly provided plasmid p29K11. DN's work on this project was supported by a Russian Science Support Foundation award.
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