Identification of the PstII cleavage loci Sequencing was performed using Sequenase 2.0 (USB). All primers were supplied by Sigma Genosys: 50-GCCTACAAT- CCATGCCAACC-30 was used for site 1 and 50-TCGCACCT- GACGTCTAAGAA-30 was used for sites 2, 3 and 4. Plasmid DNA was alkali denatured and then precipitated. The primer extension reaction contained 1.2 pmol denatured DNA and 2 pmol primer in NEB 4. The primer was annealed by heating to 90 C and cooling to 20 C for 15 min. Extension was started by addition of 2 nmol dNTPs, 1.68 nmol [a-32P]ATP and 10 U Klenow polymerase (Fermentas). Reactions were incubated at 37 C for 15 min then heat inactivated at 75 C for 20 min. PstII mixture (15.5 pmoles) and 4 mM ATP were added, and the reactions incubated for 30 min at 37 C, and then heat inac- tivated at 67 C. The reactions were then split into two and one-half treated with 2 nmol dNTPs and 1 U Klenow poly- merase at 20 C for 10 min. Reactions were stopped with the Sequenase loading buffer. Sequencing reactions were run alongside the primer extension reaction on a denaturing PAGE [6% (v/v) polyacrylamide]. Images were captured using a Molecular Dynamics Typhoon 9200 PhosphorImager. Coupled NTPase assay Measurement of NTPase activity was carried out using a coupled assay according to Kiianitsa et al. (27). Reactions (200 ml) contained 129 nM PstII mixture, 1 nM plasmid DNA (pAT153 or pSKFoK1), 4.40 U lactate dehydrogenase (Sigma-Aldrich), 2.91 U pyruvate kinase (Sigma–Aldrich), 200 mM NADH and 200 mM phosphenolpyruvate in NEB 4 supplemented with the appropriate NTP as indicated in the text. Reactions were allowed to equilibrate for 30 min to remove contaminating NDP, before addition of PstII. The absorbance at 340 nM was measured continuously for 1 h at 37 C in a VERSAmax 96-well plate reader3 (Molecular Devices). Linear ﬁts were obtained using the accompanying software (SOFTmax Pro) and the rate on the speciﬁc DNA (pAT153) corrected for the background rate on the non-speciﬁc DNA (pSKFokI). RESULTS Isolation of the PstII RM system The PstII RM system was discovered when a plasmid library of P.stuartii 164 DNA was screened in E.coli for clones that restricted the growth of bacteriophage. Restricting cells can proliferate in the presence of certain phage, whereas non- restricting cells succumb to infection and die (24). E.coli cells harbouring the library were exposed to a restriction- sensitive derivative of the virulent, T4 bacteriophage (Materi- als and Methods). Two kinds of restricting clones survived. One carried a 4.0 kb fragment that encoded the Type II RM system PstI (24) and the other carried a 6.1 kb fragment that encoded a different RM system, the existence of which was hitherto unknown. We designated this second system PstII (1). The two restricting systems were shown to differ by biochem- ical and phenotypic tests. Cell extracts of each were prepared and assayed for restriction endonuclease activity. Extracts of PstI-clones contained abundant endonuclease activity that pro- duced DNA-digestion patterns characteristic of that enzyme. Nucleic Acids Research, 2005, Vol. 33, No. 15 4779 Table 1. Approximate efficiencies of plating (eop) of phage l and F80 derivatives on E.coli harbouring cloned RM systems Phage Modification Efficiency of plating Host restriction system l CI- l vir l vir l vir F80 CI- F80 CI- F80 CI- None None PstI PstII None PstI PstII Control 1 1 1 1 1 1 1 PstI 10 5 10 5 1 10 5 10 5 1 10 5 PstII 10 5 10 6 10 6 1 10 6 10 6 1 EcoRI 10 5 10 4 Phage grow with an eop of one on cells harbouring no RM system (control). Unmodified phage are severely restricted (grow with much-reduced eop) on cells carrying either the PstI or PstII systems from P.stuartii, or the native EcoRI system. Rare phage that escape restriction acquire DNA-modification that pro- tects them from subsequent restriction by that host. PstI-modified phage con- tinue to be restricted by PstII, however, and PstII-modified phage continue to be restricted by PstI, indicating that the protective modification is system-specific, and that the specificities of the two systems are therefore different. Extracts of PstII-clones, in contrast, produced barely detect- able banding patterns that deﬁed immediate identiﬁcation. However, cells of both types restricted lambdoid phages efﬁciently (Table 1), implying that both RM systems were expressed and active in vivo. Rare plaques from phages that had escaped restriction were isolated from cells of both types, and propagated on the same cells to prepare phage-stocks bearing system-speciﬁc modiﬁcations. PstI-modiﬁed phage, no longer restricted by PstI-containing cells, continued to be restricted by PstII. Conversely, PstII-modiﬁed phage, no longer restricted by PstII, continued to be restricted by PstI (Table 1). These results implied that both systems were complete and could modify as well as restrict, and that the sequence-speciﬁcities of the PstI and PstII systems were distinct. Sequence and analysis of the PstII RM system The nucleotide sequence of the 6.1 kb fragment was determ- ined (GenBank accession no. DQ143901). The PstII system occupies the central 4.5 kb of this sequence and comprises two adjacent, similarly oriented genes, pstIIM (1685 bp) and pstIIR (2858 bp) coding for the Mod (561 amino acid) and Res (952 amino acid) subunits of what sequence comparisons suggested was an RM system of the Type III class (1). In addition to the PstII operon this fragment also contains one additional ORF encoding a putative 182 amino acid polypeptide (data not shown): this was not readily identiﬁed and was not investig- ated further. Type III RM operons consist of two genes, mod and res, which lie closely adjacent to each other and are transcribed in the same direction with mod preceeding res. The genes encode two polypeptide subunits, Mod and Res, respectively (1–4). Mod.PstII has amino acid motifs [Figure 1, (28)] and predicted secondary structural features (data not shown) characteristic of a b-group methyltransferase (29). These include: Motif I, which is likely to be involved in binding of the methyl donor S-adensoyl methionine (AdoMet); Motif IV, which is likely to be involved in the catalysis of DNA methylation at the exocyclic N6 of adenine; and a target recognition domain (TRD), which is likely to recognize a