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 the IncI1 transmissive plasmid R64 ArdA protein

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. 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 nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.     

Mutational Analysis of the Conserved Motif of the ArdA Antirestriction Protein Encoded by Self-transmissible IncI Plasmid ColIb-P9

A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD-143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plasmids and specifically inhibit type I restriction–modification systems. Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity. Hydrophobic L130, L131, and V138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D127, D150, and D154 neighboring the antirestriction motif substituted with V. Four substitutions (L130E, L131E, V138E, and D143V) substantially (25–1000 times) reduced the ArdA activity. The other substitutions within or beyond the motif had no appreciable effect. Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function. Thus, the antirestriction role of ArdA is indeed due to its conserved motif.     

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.     

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).     

Mutational analysis of the conserved motif of the Arda anti-restriction protein encoded by self-transmissible IncI plasmid ColIb-p9

A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD-143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plasmids and specifically inhibit type I restriction-modification systems. Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity. Hydrophobic L130, L131, and V138 were substituted with negatively charged E; negatively charged D133, E136, and D143 substituted with hydrophobic V; and D127, D150, and D154 neighboring the antirestriction motif substituted with V. Four substitutions (L130E, L131E, V138E, and D143V) substantially (25-1000 times) reduced the ArdA activity. The other substitutions within or beyond the motif had no appreciable effect. Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function. Thus, the antirestriction role of ArdA is indeed due to its conserved motif.     

ArdA proteins from different mobile genetic elements can bind to the EcoKI Type I DNA methyltransferase of E. coli K12

Anti-restriction and anti-modification (anti-RM) is the ability to prevent cleavage by DNA restriction–modification (RM) systems of foreign DNA entering a new bacterial host. The evolutionary consequence of anti-RM is the enhanced dissemination of mobile genetic elements. Homologues of ArdA anti-RM proteins are encoded by genes present in many mobile genetic elements such as conjugative plasmids and transposons within bacterial genomes. The ArdA proteins cause anti-RM by mimicking the DNA structure bound by Type I RM enzymes. We have investigated ArdA proteins from the genomes of Enterococcus faecalis V583, Staphylococcus aureus Mu50 and Bacteroides fragilis NCTC 9343, and compared them to the ArdA protein expressed by the conjugative transposon Tn916. We find that despite having very different structural stability and secondary structure content, they can all bind to the EcoKI methyltransferase, a core component of the EcoKI Type I RM system. This finding indicates that the less structured ArdA proteins become fully folded upon binding. The ability of ArdA from diverse mobile elements to inhibit Type I RM systems from other bacteria suggests that they are an advantage for transfer not only between closely-related bacteria but also between more distantly related bacterial species.     

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.     

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 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.     

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.     

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.     

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.     

The Orf18 gene product from conjugative transposon Tn916 is an ArdA antirestriction protein that inhibits type I DNA restriction-modification systems

Gene orf18, which is situated within the intercellular transposition region of the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA A) protein. Conjugative transposons are generally resistant to DNA restriction upon transfer to a new host. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli, and the recombinant ArdA protein was purified to homogeneity. The protein appears to exist as a dimer at nanomolar concentrations but can form larger assemblies at micromolar concentrations. R/M assays revealed that ArdA can efficiently inhibit R/M by all four major classes of Type I R/M enzymes both in vivo and in vitro. These R/M systems are present in over 50% of sequenced prokaryotic genomes. Our results suggest that ArdA can overcome the restriction barrier following conjugation and so helps increase the spread of antibiotic resistance genes by horizontal gene transfer.     

Plasmid R16 ArdA protein preferentially targets restriction activity of the type I restriction-modification system EcoKI

The ArdA antirestriction protein of the IncB plasmid R16 selectively inhibited the restriction activity of EcoKI, leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The results are consistent with the hypothesis that ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction in the recipient bacterium and yet acquire cognate modification.     

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.     

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance

The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha–beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.     

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.     

DNA mimicry by proteins as effective mechanism for regulation of activity of DNA-dependent enzymes

Modern concepts on mechanisms of DNA-dependent enzyme regulation involving specific DNA-mimicking proteins are considered. There are proteins that share structural resemblance with DNA duplexes. These include inhibitors of type I restriction-modification enzymes (Ocr and ArdA), inhibitors of DNA gyrase MfpA and QnrABS, etc. We describe here structural features of these proteins and mechanisms responsible for their interaction with DNA-dependent enzymes and then discuss perspectives of use of DNA-mimicking proteins in analysis of replication, repair, recombination, mechanisms underlying resistance to antibiotics, and also fields of applied biotechnology.