Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9

Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA--A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form--were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90-99%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R2M2S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R2M2S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.     

Antirestriction Activity of ArdA Encoded by the IncI1 Transmissive Plasmid R64

The transmissive plasmid R64 (IncI1) performs an antirestriction function, reducing the efficiency of EcoKI-dependent restriction in Escherichia coli K12 cells approximately fivefold. The R64 ardA gene has been cloned and sequenced. The ArdA proteins specifically inhibit type I restriction–modification enzymes. R64 ArdA is highly homologous to ColIb-P9 ArdA: only 4 out of 166 amino acid residues differ. While ColIb-P9 inhibits both endonuclease and methylase activities of the type I restriction–modification enzyme EcoKI (R₂M₂S), R64 ArdA inhibits only its endonuclease activity. It has been assumed that R64 ArdA suppresses the binding of unmodified DNA with the R subunit, which is responsible for DNA translocation and cleavage. ColIb-P9 ArdA suppresses DNA binding not only with the R, but also with the S subunit, which contacts the sK site containing target adenines. The binding of ArdA with the specific site inhibits both endonuclease and methylase activities; the binding of ArdA with the nonspecific site of the R subunit inhibits only the endonuclease activity ofEcoKI (R₂M₂S).     

Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9

Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA—A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form—were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R₂M₂S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R₂M₂S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.     

Role of "anti-restriction" motif in functional activity of anti-restriction protein ArdA pKM101 (incN)

A number of mutant forms of the antirestriction protein ArdA encoded by the ardA gene located in a transmissive IncN plasmid pKM101 have been constructed. Proteins belonging to the Ard family are specific inhibitors of type I restriction--modification enzymes. Single mutational substitutions of negatively charged amino acid residues located in the "antirestriction motif" with hydrophobic alanine, E134A, E137A, D144A, or a double substitution E134A, E137A do not affect the antirestriction activity (Ard) of ArdA but almost completely abolish the antimodification activity (Amd). Mutational substitutions F107D and A110D in the assumed interface ArdA, which determines contact between monomers in the active dimer (Ard)2, cause an approximately 100-fold decrease in the antirestriction protein activity. It is hypothesized that the ArdA protein forms two complexes with the type I restriction--modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with a nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonucleases. The association of ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.     

Comparative analysis of anti-restriction activities of ArdA (ColIb-P9) and Ocr (T7) proteins

Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3(ocr) genes were cloned in pUC18 vector. Both ArdA (ColIb-P9) and Ocr (T7) proteins inhibit both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. ColIb-P9 ardA, T7 0.3(ocr), and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltetO-1) promoter, which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltetO-1) promoter can be regulated over an up to 5000-fold range by supplying anhydrotetracycline to the E. coli MG1655Z1 tetR(+) cells. Effectiveness of the anti-restriction activity of the ArdA and Ocr proteins depended on the intracellular concentration. It is shown that the dissociation constants K(d) for ArdA and Ocr proteins with EcoKI enzyme differ 1700-fold: K(d) (Ocr) = 10(-10) M, K(d) (ArdA) = 1.7.10(-7) M.     

IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions.

The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.     

Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system

Many promiscuous plasmids encode the antirestriction proteins ArdA (alleviation of restriction of DNA) that specifically affect the restriction activity of heterooligomeric type I restriction-modification (R-M) systems in Escherichia coli cells. In addition, a lot of the putative ardA genes encoded by plasmids and bacterial chromosomes are found as a result of sequencing of complete genomic sequences, suggesting that ArdA proteins and type I R-M systems that seem to be widespread among bacteria may be involved in the regulation of gene transfer among bacterial genomes. Here, the mechanism of antirestriction action of ArdA encoded by IncI plasmid ColIb-P9 has been investigated in comparison with that of well-studied T7 phage-encoded antirestriction protein Ocr using the mutational analysis, retardation assay and His-tag affinity chromatography. Like Ocr, ArdA protein was shown to be able to efficiently interact with EcoKI R-M complex and affect its in vivo and in vitro restriction activity by preventing its interaction with specific DNA. However, unlike Ocr, ArdA protein has a low binding affinity to EcoKI Mtase and the additional C-terminal tail region (VF-motif) is needed for ArdA to efficiently interact with the type I R-M enzymes. It seems likely that this ArdA feature is a basis for its ability to discriminate between activities of EcoKI Mtase (modification) and complete R-M system (restriction) which may interact with unmodified DNA in the cells independently. These findings suggest that ArdA may provide a very effective and delicate control for the restriction and modification activities of type I systems and its ability to discriminate against DNA restriction in favour of the specific modification of DNA may give some advantage for efficient transmission of the ardA-encoding promiscuous plasmids among different bacterial populations.     

Role of “Antirestriction” Motif in Functional Activity of Antirestriction Protein ArdA pKM101 (IncN)

A number of mutant forms of the antirestriction protein ArdA encoded by theardA gene located in a transmissive IncN plasmid pKM101 have been constructed. Proteins belonging to the Ard family are specific inhibitors of type I restriction–modification enzymes. Single mutational substitutions of negatively charged amino acid residues located in the antirestriction motif with hydrophobic alanine, E134A, E137A, D144A, or a double substitution E134A, E137A do not affect the antirestriction activity (Ard) of ArdA but almost completely abolish the antimodification activity (Amd). Mutational substitutions F107D and A110D in the assumed interface ArdA, which determines contact between monomers in the active dimer (Ard)₂, cause an approximately 100-fold decrease in the antirestriction protein activity. It is hypothesized that the ArdA protein forms two complexes with the type I restriction–modification enzyme (R₂M₂S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with a nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonucleases. The association of ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R₂M₂S enzyme.     

Antimodification activity of the ArdA and Ocr proteins

The ArdA and Ocr antirestriction proteins, whose genes are in transmissible plasmids (ardA) and bacteriophage genomes (0.3 (ocr)), specifically inhibit type I restriction-modification enzymes. The Ocr protein (T7 bacteriophage) was shown to inhibit both restriction (endonuclease) and modification (methylase) activities of the EcoKI enzyme in a broad range of intracellular concentrations (starting from 10–20 molecules per cell). In contrast to Ocr, the ArdA protein (ColIb-P9 transmissible plasmid) inhibited both of the EcoKI activities only at high intracellular concentrations (30000–40000 molecules per cell). When the ArdA concentration was several fold lower, only endonuclease activity of EcoKI was inhibited. It was assumed that a poorer ArdA ability to inhibit EcoKI modification activity is related to the substantial difference in life cycle between transmissible plasmids (symbiosis with the bacterial cell) and bacteriophages (infection and lysis of bacteria). The Ocr and ArdA mutants that inhibited exclusively endonuclease activity of EcoKI were obtained. Antirestriction proteins incapable of homodimerization were assumed to inhibit only endonuclease activity of type I restriction-modification enzymes.     

Comparative analysis of antirestriction activity of the ArdA and ArdB proteins encoded by genes of the R64 transmissible plasmid (IncI1)

Antirestriction proteins ArdA and ArdB are specific inhibitors of type I restriction-modification enzymes. The ardA and yfeB (ardB) genes were cloned from the transmissible plasmid R64 in the pUC18 and pZE21 vectors. The R64 ArdA and ArdB proteins were shown to inhibit only restriction activity of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. In contrast to ArdA, ArdB inhibited EcoKI restriction activity only at a high intracellular concentration. Antirestriction activity of ArdB did not depend on the ClpXP protease. The yfeB (ardB) gene of the R64 plasmid is transcribed from a weak promoter located upstream of yfeA.     

Antirestriction proteins ArdA and Ocr as efficient inhibitors of type I restriction-modification enzymes

Genes encoding antirestriction proteins are found in transmissble plasmids (ardABC) and bacteriophage genomes (ocr, darA). Antirestriction proteins inhibit type I restriction-modification enzymes and thus protect the unmodified plasmid or phage DNA from degradation. Antirestriction proteins belong to the family of DNA-mimicry proteins, whose spatial structure mimics the B-form of DNA. Based on an analysis of the mutant forms of ArdA and Ocr obtained by site-directed mutagenesis and the native form of ArdA that specifically inhibit type I restriction enzymes but do not affect their methylase activity, a model is proposed to describe the complex formation between an antirestriction protein and a type I restriction-modification enzyme (R₂M₂S): antirestriction proteins can displace a DNA strand from its binding sites in the S subunit (which contacts a specific site on DNA) and in the R subunit (which translocates the DNA strand and cleaves it). Antirestriction and antimodification activities of ArdA and Ocr as a function of ardA and ocr expression levels were studied by cloning the genes under a strictly regulated promoter.     

Stepwise cloning and molecular characterization of the HgiDI restriction-modification system from Herpetosiphon giganteus Hpa2.

The restriction-modification system HgiDI from Herpetosiphon giganteus strain Hpa2 has been cloned in E. coli in a two-step procedure. Selection of the methyltransferase (M.HgiDI) gene in vitro was performed using the heterologous restriction endonuclease AhaII, an isoschizomer of Acyl and HgiDI (GRCGYC). Cloning of the complete HgiDI endonuclease (R.HgiDI) gene could only be achieved in recipient cells harbouring a recombinant plasmid, which was expressing the corresponding methyltransferase and could thereby prevent the host from self-destruction of its genetic material. The HgiDI restriction-modification system was sequenced and functionally correlated with two open reading frames of 309 (M) and 359 (R) codons. In homology studies M.HgiDI showed significant similarities to 20 other m5C-methyltransferases and turned out to be the most compact enzyme of this group described so far. Initial attempts for overexpression of M.HgiDI and partial purification of R.HgiDI have been successful.     

Structural and functional analysis of the symmetrical Type I restriction endonuclease R.EcoR124I(NT)

Type I restriction-modification (RM) systems are comprised of two multi-subunit enzymes, the methyltransferase (～160 kDa), responsible for methylation of DNA, and the restriction endonuclease (～400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits. An engineered RM system, EcoR124I(NT), based on the N-terminal domain of the specificity subunit of EcoR124I was constructed that recognises the symmetrical sequence GAAN(7)TTC and is active as a methyltransferase. Here, we investigate the restriction endonuclease activity of R. EcoR124I(NT)in vitro and the subunit assembly of the multi-subunit enzyme. Finally, using small-angle neutron scattering and selective deuteration, we present a low-resolution structural model of the endonuclease and locate the motor subunits within the multi-subunit enzyme. We show that the covalent linkage between the two target recognition domains of the specificity subunit is not required for subunit assembly or enzyme activity, and discuss the implications for the evolution of Type I enzymes.     

Detection of primase specified by IncB plasmid R864a.

Plasmid R864a (IncB) contains nucleotide sequences homologous with the sog primase determinant of IncI alpha plasmid ColIb-P9. Extracts of Escherichia coli carrying a mutant R864a derepressed for transfer functions showed enhanced primase activity, and contained a large polypeptide identical in size (apparent Mr = 220,000) to the IncI alpha sog gene product.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

The effect ofrecA mutation on the expression ofEcoKI andEcoR124Ihsd genes cloned in a multicopy plasmid

Type I restriction-modification (R-M) endonucleases are composed of three subunits—HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of clonedEcoKI andEcoR124Ihsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wthsd genes in a multicopy plasmid inEscherichia coli recA ⁺ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the clonedhsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation inE. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.     

In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants

The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.     

Analysis of the subunit assembly of the typeIC restriction-modification enzyme EcoR124I.

Type I restriction-modification (R-M) enzymes are composed of three different subunits, of which HsdS determines DNA specificity, HsdM is responsible for DNA methylation and HsdR is required for restriction. The HsdM and HsdS subunits can also form an independent DNA methyltransferase with a subunit stoichiometry of M2S1. We found that the purified Eco R124I R-M enzyme was a mixture of two species as detected by the presence of two differently migrating specific DNA-protein complexes in a gel retardation assay. An analysis of protein subunits isolated from the complexes indicated that the larger species had a stoichiometry of R2M2S1and the smaller species had a stoichiometry of R1M2S1. In vitro analysis of subunit assembly revealed that while binding of the first HsdR subunit to the M2S1complex was very tight, the second HsdR subunit was bound weakly and it dissociated from the R1M2S1complex with an apparent K d of approximately 2.4 x 10(-7) M. Functional assays have shown that only the R2M2S1complex is capable of DNA cleavage, however, the R1M2S1complex retains ATPase activity. The relevance of this situation is discussed in terms of the regulation of restriction activity in vivo upon conjugative transfer of a plasmid-born R-M system into an unmodified host cell.     

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.