prediction and zero ﬁlling prior to apodisation with a 5% shifted mixed Gaussian/exponential function. NOE peak heights were taken from the Gaussian ﬁts of the frequency-domain data. The NOE was calculated as NOE = Ion/Ioﬀ where Ion and Ioﬀ are the peak heights with and without 1H pre-saturation, respectively. T1, T2 and NOE data were ﬁtted using ‘Quadric_diﬀusion’, (AG Palmer, Columbia University, NY, USA, http://biochem istry.hs.columbia.edu/labs/palmer/software/quadric.html) to obtain the rotational diﬀusion tensor. Model-free analysis of the relaxation data was performed using Tensor2 (51). For this analysis a subset of relaxation data was selected using criteria deﬁned by Tjandra et al. (52). The relative moments of inertia were calculated with ‘pdbinertia’ (http://biochemistry.hs.columbia.edu/labs/ palmer/software/pdbinertia.html) and the average rotational correlation time (tc) calculated with r2r1_tm (http://biochemistry.hs.columbia.edu/labs/ palmer/software/r2r1_tm.html). The complete set of coordinates for the structure has been deposited at the Protein Data Bank (PDB) (53) with accession code 2 kmg and in the Biological magnetic resonance bank with code 16428. Bioinformatics analysis of sequence and structure Secondary structure was identiﬁed using STRIDE (54). Fold topology analysis was conducted using HERA (55) under PROMOTIF v2.0 (56) and schematically redrawn using TopDraw (57). The B. pertussis KlcA sequence was subjected to a BLAST search (58) against the PDB and to fold recognition server PHYRE (59) to check if there was any clear or remote homology to an existing structure or fold at the sequence level. Additionally, the 3D-coordinates of the KlcA structure were compared against 3D-fold and structure libraries using DALI (60) and SSM (61). Sequences related to B. pertussis KlcA (UniProt Accession no: Q08L07) were obtained using a BLASTP search (58) against the UniProt sequence database (62), via the NPS@ server (63). A total of 182 sequences with an E-value of less than 10 6 were obtained and this list was made non-redundant initially by removing 62 identi- cal sequences. The remaining 120 KlcA-like sequences were retrieved and a multiple sequence alignment was generated using the program PROMALS3D (64). This set of sequences was rendered further non-redundant by removing all 90% pairwise identical sequences in the alignment using the Expasy server (http://www.expasy .ch/tools/redundancy/). A total of 44 non-redundant divergent sequences were thus obtained. Upon analysis, one sequence that lacked the C-terminal helix 5 was dis- carded and three other sequences, which possessed N-terminal extensions, were trimmed. The remaining 43-aligned sequences were used as input for Consurf (65) (http://consurf.tau.ac.il/) to map sequence conservation on the surface of the protein. The electrostatic surface potential representation was calculated using GRASP (66). The lipophilic surface rendition was generated using MOLCAD (67) under SYBYL (Tripos Associates, Nucleic Acids Research, 2010, Vol. 38, No. 5 1727 St Louis, MO, USA). Solvent-accessibility calculations were performed using GETAREA (68). RESULTS The ArdB proteins from the E. coli pathogenicity island, the CP4-6 prophage, the CP4-57 prophage and L. pneumophila will be referred to as ArdBCFT, ArdBYAF, ArdBYFJ and ArdBLPP, respectively. The KlcA proteins from B. pertussis and A. denitriﬁcans will be referred to as KlcA136 and KlcAADE, respectively. The names ArdB and KlcA without a subscript are to be understood as referring to all of the ArdB or KlcA studied. Overexpression of klcA is not lethal to E. coli It has been shown that unregulated expression of the kil genes leads to killing of the host cells. The cells become elongated with distorted outer membranes and macromolecular synthesis ceases (69). Previous studies of the kilC operon in plasmid RK2 showed that klcA was responsible for the lethality of E. coli host cells in the absence of korA and korC (20). However, we were able to overexpress the klcA136 gene in a pET vector in the absence of kor determinants with no apparent harmful eﬀects. Furthermore, in vivo anti-restriction experiments performed on the klcA from RK2 failed to identify any signs of anti-restriction function (20), whereas the klcA136 and klcAADE showed eﬃcient anti-restriction activity against all four families of Type I restriction systems as described below. In vivo activity of ardB and klcA genes against the four families of Type I RM systems The activity of anti-restriction genes can be tested by determining how many phages successfully infect a bacte- rial lawn in the presence or absence of host RM systems when the bacterium also contains a plasmid encoding the anti-restriction protein. All of the ardB and klcA genes except ardBLPP showed signiﬁcant levels of anti-restriction (>10-fold enhancement in the number of phage infec- tions) despite their low levels of expression in the absence of induction, Table 2. The poor activity of the ardBLPP and klcAADE 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. However, in contrast to ocr and ArdA, anti-modiﬁcation by ArdB was generally very weak (<3-fold and negligible given the limitations of the assay), Table 2. In other words, the phages that escaped restriction were easily modiﬁed by the methylation activity of the RM system and ArdB had little if any inhibitory eﬀect on methylation. In three cases, some inhibition of methylation was observed; ArdBCFT and ArdBYFJ slightly inhibited methylation by the Type ID RM system and ArdBYAF slightly inhibited methylation by the Type IC RM system. These results show a clear diﬀerence