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

Abstract

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)

Genome Modification in Enterococcus faecalis OG1RF Assessed by Bisulfite Sequencing and Single-Molecule Real-Time Sequencing

“…Activities of the EfaRFI system against singlestranded DNA and hemimethylated DNA are as yet unknown, and further studies will be required to assess its spectrum of biochemical activities. We note also that pCF10 harbors genes for a predicted ArdA protein within a Tn925 element and that ArdA proteins inhibit type I REases by acting as a DNA mimic (71)(72)(73)(74). Whether the pCF10 ArdA has any impact on EfaRFI, a type II system, remains to be determined.…”

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

“…Ultracentrifugation has previously defined these as monomer and dimer forms of ArdA [20]. Furthermore, the size exclusion data show that ArdA proteins can interact with the core MTase of EcoKI and the M 1 S 1 partially assembled core.…”

“…ArdA genes are expressed from a novel single-stranded promoter as soon as the conjugative plasmid or transposon enters a new host [7,19] . The predicted ArdA proteins show considerable sequence variation and the genes often encode long N-terminal and/or C-terminal extensions [20] . These differences between Ocr and ArdA may relate to the different life styles of the parent MGE, namely a lytic phage versus a conjugative plasmid or transposon.…”

Diversity and Evolution of the Tn 5801-tet (M)-Like Integrative and Conjugative Elements among Enterococcus, Streptococcus, and Staphylococcus

“…In this respect, we should take into account therapeutic strategies and relevant bacterial features that could have facilitated the selective acquisition of certain elements by particular clones. For example, anti-restriction-modification (anti-RM) occurring in Tn916-like elements often influences selective horizontal gene transfer between staphylococcal populations and other species of Firmicutes (43,44). Putative anti-RM detected in Tn5801 (orf12) acts against staphylococcal type I RM systems which are present in S. aureus CC5 and CC8 (the backgrounds in which Tn5801 seems to have initially been detected in the late 1950s) (3,15) and ST239 (an emergent hybrid lineage of ST8 and ST30) (10,17,45).…”