1724 Nucleic Acids Research, 2010, Vol. 38, No. 5 evolutionary pressure on mobile genetic elements to evolve anti-restriction countermeasures encoded by the foreign DNA (2,3,7). One such measure is the use of anti-restriction proteins to modify or inactivate the RM system. These are frequently targeted against Type I RM systems and operate in a variety of diﬀerent manners. The simplest anti-restriction proteins mimic the structure of DNA and include the proteins ocr from phage T7 and ArdA from plasmids and conjugative transposons (8,9). The ocr protein from phage T7 (9,10) and the ArdA proteins from conjugative plasmids and conjugative transposons (3,8,11–15) have been the most intensively studied of the DNA mimics. These inactivate the host’s Type I RM enzyme by binding in the substrate (DNA) binding groove (16,17). The structure of these proteins shows they mimic DNA both in size and surface charge distribution and have high aﬃnity for binding to the RM enzyme. Another group are single-strand DNA binding proteins and represented by the ArdC protein (7,18). Binding to the single-stranded DNA intermediate formed during conjugation seems to lead to anti- restriction though why this occurs is unclear as the Type I RM systems act on double-stranded DNA (11,18). The ArdB proteins (7,11,18) and their close homologues the KlcA proteins (19,20) are the subject of this study. In annotated sequence databases, the ArdB proteins appear to be mostly conﬁned to the proteobacteria. For instance, Escherichia coli K12 W3110 contains two prophages on its chromosome, CP4-6 and CP4-57, encoding ardB homologues termed yafX and yfjX, respec- tively (21). Comparison of the predicted products of these two genes with sequence databases indicates that they are highly homologous to the ArdB protein from the IncN incompatibility group conjugative plasmid pKM101 (11). Similarly, another putative ardB, ORF14, is encoded by the second pathogenicity island, PAI IICFT073, from the uropathogenic E. coli CFT073 (22,23). Putative homologues have also been identiﬁed in Legionella pneumophila (24) and Achromobacter denitriﬁcans (25). The ArdB proteins are small in size with an acidic nature, which is similar to ocr and ArdA. The anti- restriction function for ardB from pKM101 has been demonstrated but it was also observed that it did not inhibit modiﬁcation (11) in contrast to the ocr and ArdA DNA mimics. This inability to block modiﬁcation occurred despite the presence of the short ‘anti-restriction amino acid motif’ identiﬁed in ocr, ArdA and ArdB (7,26). The recent atomic structure of ArdA indicates that this motif has a structural role in maintaining the fold of ArdA rather than a direct role in inhibiting the RM enzyme (8). Therefore, the presence of this motif in ArdB does not necessarily imply a functional role. klcA is one of three genes found on the kilC operon, one of four kil loci (20) found on IncP plasmids. Expression of kil genes can be lethal to E. coli and is regulated by the kor genes. korA and korC control klcA on the broad host range plasmid RK2. klcA from RK2 showed 31% identity and 56% similarity to ardB from pKM101. Plasmids of the incompatibility group P are found in a wide range of Gram-negative bacteria (27). IncP-1b members usually carry multiple antibiotic resistance determinants and genes for the degradation of xenobiotic compounds (28). An exception is the IncP-1b plasmid pA1 from Sphingomonas sp. A1 that does not contain any of the typical accessory genes of IncP-1bplasmids (29). Similarly, pBP136, isolated from Bordetella pertussis in a fatal case of whooping cough (19) was found to contain no accessory genes for antibi- otic resistance, remnants of insertion sequences or transposons. It contains two regions involved in plasmid conjugation and a third region involved in plasmid repli- cation, central control, stable inheritance and partitioning. This region contained a klcA gene encoding a protein with 71% identity to KlcA from RK2. Sequence alignment revealed that KlcA from pBP136 is also signiﬁcantly related to the anti-restriction ArdB proteins (19) showing, e.g. 30% identity and 46% similarity to the 16.5 kDa ArdB from the IncN plasmid pKM101 (11). The signiﬁcant homology of KlcA to ArdB implied a possible function of KlcA as an anti-restriction protein. However, klcA from the IncP-1a plasmid RK2 showed no anti-restriction activity against the archetypal Type I RM system EcoKI from E. coli (20). Here, we demonstrate that four ardB and two klcA display an anti-restriction function in vivo against Type I RM systems but contrary to expectations, we ﬁnd that in vitro, the puriﬁed ArdB proteins are unable to prevent the normal restriction endonuclease activity of the EcoKI Type I RM enzyme indicating an anti-restriction mecha- nism distinct from the DNA mimicry used by the ArdA anti-restriction proteins (8,12,13). We present the NMR-derived atomic resolution structure of the full length, 142 amino acid, KlcA protein from plasmid pBP136 isolated from B. pertussis and show that it has a novel fold and clearly is not a DNA structural mimic. METHODS Strains, plasmids and genes Escherichia coli JM109 was used as a general cloning strain (Promega Madison, WI, USA). The expression strain E. coli BL21(DE3) was purchased from Invitrogen (Groningen, The Netherlands). The E. coli strains used for assessing restriction activity in vivo comprise NM1261 (r m ), NM1049 (restriction system Type IA), NK354 (Type IB), NK402 (Type IC), NM1009 (Type ID) and were a kind gift of Professor Noreen E. Murray (School of Biology, University of Edinburgh, UK). NM1041 (hsd+ clpX ) was again obtained from Noreen Murray (30). The NK and NM series were converted to DE3 lysogens as described previously (8). Four ArdB genes were identiﬁed in the public sequence database held by the National Center for Biotechnology Information (Bethesda, MD, USA), Table 1. The gene sequences were then synthesised by GeneArt (Regensburg, Germany). The codon usage pattern was optimized in order to maximize expression yield in E. coli. To facilitate cloning, an NdeI restriction site was engineered to overlap the ATG start codon of each synthetic gene. In addition, a unique HindIII site was also included just downstream of the stop codon.