1728 Nucleic Acids Research, 2010, Vol. 38, No. 5 Table 2. Eﬀect of expression of six diﬀerent ardB and klcA on the RM of bacteriophage v by four diﬀerent Type I RM systems, each representing one of the four families (IA–ID) of Type I RM systems Gene Chromosomal restriction system in host E. coli NM1049 Type IA EcoKI NK354 Type IB EcoAI NK402 Type IC EcoR124I NM1009 Type ID StySBLI Anti-restriction Anti-modiﬁcation Anti-restriction Anti-modiﬁcation Anti-restriction Anti-modiﬁcation Anti-restriction Anti-modiﬁcation ardBCFT ardBYAF ardBYFJ klcAADE klcA136 ardBLPP Vector alone 710 9250 6160 1140 2430 270 1 2.0 1.4 1.2 1.6 1.4 1.1 1 54 27 10 14 76 17 1 1.8 1.6 1.1 1.2 1.1 1.1 2 199 7510 251 13 628 1.9 1 1.3 10.6 1.0 1.9 3.1 1.4 2 1174 1080 1650 37 969 132 1 6.0 1.5 18.6 1.2 1.4 1.4 1 The host E. coli contained a chromosomal copy of the RM system and an uninduced expression plasmid expressing ardB or klcA at low level. An anti-restriction or anti-modiﬁcation value >2 was taken to indicate anti-restriction activity in vivo. Errors in the anti-restriction and anti-modiﬁcation values are ± 25%. between the activities of ArdB and KlcA compared to the DNA mimics, ocr and ArdA, suggesting that they use dif- ferent mechanisms for interfering with the Type I RM systems. KlcA136 anti-restriction function does not involve the ClpXP protease It has been shown that ClpXP-dependent regulation of the EcoKI endonuclease activity enables bacteria that acquire unmodiﬁed chromosomal target sequences to survive. When cells are under stress after prolonged exposure to treatments that damage DNA, such as exposure to 2-AP, the ClpXP-dependent pathway operates to prevent EcoKI from cleaving the chromosome (71). The restriction subunit of the nuclease (HsdR) is degraded in the presence of ClpXP (71). As described later, we did not observe any direct interaction of KlcA136 with the EcoKI in vitro; therefore we tested the hypothesis that the anti-restriction activity of KlcA136 was dependent on the ClpXP pathway. A clpX mutant, NM1041(DE3), was plated in the presence and absence of 80 mg/ml 2-AP, Figure 1. The clpX mutant was also transformed with the plasmid, pET20b-klcA136, expressing klcA136. As expected, the clpX strain with no anti-restriction deter- minant present, grew well in the absence of 2-AP, but no growth was observed in the presence of 2-AP. In contrast, the clpX strain carrying klcA136 grew equally well in both the presence and absence of 2-AP. This ﬁnding indicates that KlcA136 is able to block EcoKI restriction without involving the ClpXP protease. We assume that this result holds for ArdB and KlcA in general. Solution state behaviour of ArdB and KlcA proteins During puriﬁcation it was noted that ArdBYAF and ArdBCFT behaved anomalously on a large volume (60 cm 1.6 cm) Superdex 200 size-exclusion chromatog- raphy column despite showing only a single homogeneous species of the correct molecular weight on SDS–PAGE (data not shown) and mass spectrometry, Supplementary Table S1. Analysis with a small volume analytical column (25 cm 0.46 cm) revealed two peaks corresponding to a dimer and a monomer for ArdBYAF while ArdBCFT Figure 1. The eﬀect of expressing KlcA136 in a 2-AP sensitive strain of E. coli. A single colony of the ClpX strain NM1041(DE3) harbouring either the KlcA expression construct (pET20b-klcA136) or the corre- sponding vector alone (pET20b) was picked with a loop and spread onto an LB agar plate supplemented with 100 mg/ml carbenicillin, 25 mg/ ml kanamycin in the absence (A) or presence (B) of 2-AP (80 mg/ml). The plates were then incubated at 37 C for 14 h. Figure 2. An overlay of elution proﬁles for diﬀerent proteins from a small volume (25 cm 0.46 cm diameter) size exclusion chromatography column showing the variable quaternary structure of the four diﬀerent proteins in 20 mM Tris, 20 mM MES, 0.2 M NaCl, 0.1 mM EDTA, 7 mM b-mercaptoethanol, pH 6.5: ArdBCFT (black, 4 mM)), ArdBYAF (blue, 3 mM), ArdBYFJ (red, 5 mM) and KlcA136 (green, 4 mM). The proteins were detected using tryptophan ﬂuorescence emission and are arbitrarily scaled vertically. showed two peaks on the large volume column and a broad peak with shoulders on the small volume column, Figure 2. ArdBYFJ and KlcA136 showed only a single monomer peak. These results indicate that there was