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

Abstract

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.

Complete genome sequence and phenotype microarray analysis of Cronobacter sakazakii SP291: a persistent isolate cultured from a powdered infant formula production facility

“…Toxin-antitoxin pairs were noted in a previous study as most Cronobacter genomes contain a large number of them, which might be conserved, shared, or unique (Grim et al, 2013). GR-c (genome regions 891,557…912,700) contains seven interesting genes, which includes an uncharacterized protein YeeT, a NgrB protein, an ATP-dependent Clp protease, an ATP-binding subunit ClpA, a small HspC2 heat shock protein, a galactoside O-acetyltransferase-encoding gene and an anti-restriction protein KlcA which have been reported as a component part of a type I DNA restriction system (Serfiotis-Mitsa et al, 2010). A helicase protein, a glycerol dehydrogenase enzyme-encoding gene, and a DNA-cytosine methyltransferase were identified within GR-f (genome regions 3,363,441…3,392,072).…”

A novel gene,ardD, determines antirestriction activity of the non-conjugative transposon Tn5053and is located antisense within thetniAgene

“…It has been shown that the genes encoding the antirestriction proteins (ArdA, ArdB, ArdC) may be located within conjugative plasmids and conjugative transposons (Delver et al ., ; Belogurov et al ., , ; McMaahon et al ., ; Serfiotis‐Mitsa et al ., ). Here we show for the first time that a similar gene is also present within a non‐conjugative transposon (Tn 5053 ).…”

“…Conjugative plasmids and conjugative transposons contain the ardA, ardB and ardC genes, coding for antirestriction proteins. The ArdA, ArdB and ArdC proteins specifically inhibit type I restriction-modification enzymes (Delver et al, 1991;Belogurov et al, 1993Belogurov et al, , 2000Serfiotis-Mitsa et al, 2010). The ArdA proteins simultaneously inhibit restriction (endonuclease) and modification (methylase) acitivity of these enzymes (Delver et al, 1991;McMaahon et al, 2009), while the ArdB proteins inhibit only restriction activity of the enzymes (Belogurov et al, 1993;Serfiotis-Mitsa et al, 2010).…”

“…The ArdA proteins (165-170 amino acids) carry a considerable negative charge (À25: À30) and belong to the family of DNA mimic proteins, because their spatial structure is similar to the doublehelical DNA in B form (McMaahon et al, 2009). The ArdB proteins (145-153 amino acids) usually carry a small negative charge (À1: À6) and form a structure of a compact tetraeder (Serfiotis-Mitsa et al, 2010). The presence of the ardA and ardB genes helps mobile elements to overcome the restriction barriers, providing efficient 'horizontal' gene transfer between bacteria of various species and genera.…”

ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range

“…ArdA (2W82) is structurally similar to the B-form DNA this way binding type I R-M systems to avoid DNA degradation [22]. KlcA (2KMG) and ArdB (2WJ9) are composed of a single α/β domain inhibiting the endonuclease activity of Type I R-M systems by an indirect mechanism not related to the mimic of DNA structure [23]. According to our solved structure, we expect ArdC to provide a new antirestriction mechanism.…”