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

Zavilgelsky, G. B.; Kotova, V. Yu.; Расторгуев, С. М.

doi:10.1134/s0006297908080087

Cited by 20 publications

(13 citation statements)

References 11 publications

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“…Nor can the loss of antimodification activity be attributed to low protein expression levels, as these mutant proteins all expressed well. Our results obtained in vivo agree with earlier work [21,[23][24][25] where in vivo expression levels of ArdA from the ColIb-P9 plasmid were varied. These in vivo experiments showed that antirestriction was prevalent over antimodification when the concentration of ArdA was low.…”

Section: Plasmid Namesupporting

confidence: 91%

“…ORF18 ArdA appears to be able to dissociate into monomers at low concentrations in buffer solution [17], raising the possibility that the monomer form may be active in addition to the dimer form. It may even be the case that one form targets the modification activity and the other form targets the restriction activity of the RM system, as some ArdA proteins show differential effects on restriction and modification, depending on the level of expression in vivo [16,[21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

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Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

et al. 2013

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ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restrictionmodification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res 37, 4887-4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via sitedirected mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction. Structured digital abstractArdA and ArdA bind by molecular sieving (1, 2) ArdA and ArdA bind by cosedimentation in solution (1,2)

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Section: Plasmid Namesupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

et al. 2013

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“…The poor activity of the ardB LPP and klcA ADE genes were most likely due to poor expression as neither could be observed on SDS–PAGE. The effectiveness of the other proteins in inhibiting restriction matched levels observed for the ocr (9,10,15,70) and ArdA proteins (8,12,13) in vivo under similar conditions. …”

Section: Resultssupporting

confidence: 56%

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

Serfiotis-Mitsa

Herbert

Roberts

et al. 2009

Nucleic Acids Research

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Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic.

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“…This difference in the ability of ArdA to inhibit methylation of phage DNA by Type I families may reflect different binding affinities of the ArdA proteins for the Type I enzymes. At present, binding affinity is not well characterized but for the ArdA–EcoKI interaction it has been estimated that the dissociation constant, K d , is <1 nM in vitro (27) and ∼170 nM in vivo (68). This is not as strong as the interaction between Ocr and EcoKI, where the K d is ∼50 pM (69); therefore, perhaps ArdA is just not such a strong competitive inhibitor as Ocr.…”

Section: Discussionmentioning

confidence: 99%

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

McMahon

Roberts

Johnson

et al. 2009

Nucleic Acids Research

105

113

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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.

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

Cited by 20 publications

References 11 publications

Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

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

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