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Restriction activity against incoming bacteriophage and
plasmid DNA provides bacteria with a sophisticated defence mechanism against
invading 'foreign' DNA. However, evolution of the many thousands of
restriction-modification systems is only a small part of this continual battle
for survival for bacteria and bacteriophage (or plasmid). Both
bacteriophage and plasmid encoded antirestriction systems have been identified
with a variety of modes of action. The Ocr system:Ocr is encoded by gene 0.3 of bacteriophage T7 and was show to inhibit Type I restriction activity by Kruger et al (1977). The mode of action of this protein was shown to involve inhibition of the DNA-binding of the Type I R-M enzyme to the recognition sequence and Walkinshaw et al (2002) demonstrated that this was due to the protein acting as a DNA mimic. EcoKI was shown to bend DNA upon binding (Walkinshaw et al 2002) and perhaps the great significance of the mode of action of Ocr antirestriction protein is that it mimics DNA as a bent structure with the same angle of bend as that produced by EcoKI binding (Walkinshaw et al 2002). The Ard system:Ard is a plasmid-encoded alleviation of restriction protein that specifically interferes with the restriction activity of Type I R-M enzymes. This allows improved efficiency of horizontal transfer of promiscuous plasmids and as such is an important aspect of increasing antibiotic resistance. Ard protein fall into three classes - ArdA, ArdB and ArdC - on the basis of protein sequence homology and are encoded by plasmids of the IncFV, IncN and IncW plasmids (Nekrasov et al ., 2007). Ard have a conserved motif known as the anti-restriction motif (Belogurov & Delver, 1995), which has similarities to the conserved repeats of the HsdS subunit of Type I R-M enzymes suggesting a structural basis to the mode of action of Ard. A putative ArdA protein has been identified as part of the conjugative transposon Tn916 (Serfiotis-Mitsa et al 2008), which was first identified from the bacteria Enterococcus faecalis DS16 (Franke & Clewell, 1981), suggesting this system is also able to enable conjugal transfer of transposons against restriction-modification systems. Recent studies with purified ArdA, encoded by IncI plasmid ColIb-P9, (Nekrasov et al ., 2007) indicate that like Ocr, ArdA can also interact with EcoKI, inhibiting normal DNA binding. However, unlike Ocr, ArdA can "discriminate" between EcoKI MTase and REase and shows a lower binding efficiency to the MTase than that shown against the REase. This would suggest that control of restriction activity may be "finely tuned" for ArdA and provide a very specific function in vivo. The ArdA-like protein from Tn916 was found to exist as a stable dimer at nanomolar concentrations (Serfiotis-Mitsa et al 2008), but higher order assemblies were observed at micromolar concentrations. This ArdA-like protein inhibits all four families of Type I restriction-modification systems, but shows maximum anti-restriction activity against the Type IA family and maximum anti-modification activity against the Type ID system. These activities were only observed when the ArdA concentration was significantly greater than that of the MTase or ENase with an apparent stoichiometry of 1.3 ArdA monomers per MTase. There is some evidence that this ArdA may bind HsdR as well as binding the core MTase, which was simuilar to the situation with Ocr where multiple binding sites on EcoKI were observed (Atanasiu et al 2001). The Stp polypeptide:Stp is encoded by the bacteriophage T4 and is a short (~26 residue) protein, produced from one of the very early genes of the phage following injection of phage DNA into a host cell (Kaufmann et al 1986). The anti-restriction activity of this system is very complex and involves both host and phage encoded gene products. The Stp peptide has been shown to interact with a host encoded anti-codon nuclease - an enzyme that cleaves the tRNALys charged tRNA molecule, which in turn leads to cell death - this anti-codon nuclease (ACNase) exists in a latent from within the bacterial cell (otherwise it would be lethal to the cell) and this latent form involves an interaction with the host encoded Type I R-M enzyme (Amitsur et al., 1992; Penner et al., 1995).
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• © University of
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It
has been shown that the Stp peptide shifts the equilibrium between R2-
and R1-forms of the EcoR124I R-M enzyme (Figure left), with Stp
introducing a significant shift toward R1-complex formation, which is
a restriction-deficient form of the REase and explains the anti-restriction
activity of Stp (