Cloning of PstII RM system E.coli cells expressing the cloned PstII RM system were selec- ted from a genomic library of P.stuartii DNA by their ability to restrict bacteriophage as follows. Total cellular DNA of P.stuartii 164 was puriﬁed, digested with HindIII, ligated into similarly-cleaved pBR322 (20), and competent E.coli RR1 cells (16) transformed with the clones. Transformed colonies were grown overnight at 37 C on plates containing ampicillin (50 mg/ml), and collectively harvested to make the cell library (21). Aliquots of this library, containing 1 · 108 cells, were spread onto ampicillin-plates together with 1 · 109 particles of T4GT7-cyt, and incubated overnight at 37 C [T4GT7-cyt is a derivative of T4 bacteriophage that contains cytosine in its DNA in place of the glucosylated hydroxymethylcytosine present in wild-type T4. The absence of the glucosylated hydroxymethyl modiﬁcation renders the DNA sensitive to digestion by restriction enzymes in vitro, and renders the phage particles susceptible to host-controlled restriction in vivo (22,23)]. Phage-resistant colonies of two distinct morphologies arose on the plates; examples of both were picked, puriﬁed by single-colony isolation and analysed. Colonies with irregular morphology and ragged margins car- ried plasmids containing a fragment of 4.0 kb, colonies with a normal morphology and smooth margins carried plasmids containing a fragment of 6.1 kb. The former were shown to encode the PstI RM system (24), and the latter to encode the PstII RM system (see below). Clone characterization Colonies of both kinds were assayed for endogenous restric- tion endonuclease activity. Cell-extracts were prepared, seri- ally diluted, incubated for 1 h at 37 C with lambda phage DNA in NEB 3 [50 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT (pH 7.9)] and DNA products separated by agarose gel electrophoresis. E.coli clones were tested for their ability both to restrict and to modify lambdoid phages in the follow- ing way. Cell cultures were grown to saturate at 37 C in Luria– Bertani (LB) media, and 0.2 ml aliquots were mixed with 3.5 ml molten top agar and poured onto Tryptone agar plates (16). Once solidiﬁed, 10 ml drops of various phages (New England Biolabs strain collection), serially diluted from 1010 particles/ml to 104 particles/ml, were spotted onto the plate surfaces, and after overnight incubation at 37 C the plates were examined. The efﬁciency of phage plating (eop) was estimated as the ratio of the plaque count on the cells carrying the cloned RM system versus the plaque count on the same cells carrying the plasmid vector but without the RM system. Modiﬁed phage stocks were prepared by picking rare plaques that grew on the restricting cells, and propagating them on the same cells to produce high-titre lysates. DNA sequencing and analysis The nucleotide sequence of the 6.1 kb fragment was determ- ined by ‘primer-walking’ using synthetic 20–24 nt primers (New England Biolabs Organic Synthesis Division, Beverly MA). Sequencing started on each side of the HindIII site of pBR322 and proceeded into the recombinant fragment in both directions, using the nucleotide sequence obtained in each cycle to design the primer for the next. Template DNA was puriﬁed using Qiagen midi-columns and resuspended Nucleic Acids Research, 2005, Vol. 33, No. 15 4777 to 100 mg/ml in water. Sequencing reactions comprised 5 ml of template DNA (0.5 mg), 1 ml of primer (3.2 pmol) and 4 ml Amplitaq DNA polymerase FS premix (AB PRISM dye ter- minator cycle sequencing ready reaction kit; Applied Bio- systems). The reactions were prepared in 96-well plate format using a Biomek 2000 robot (Beckman) and thermo- cycled 25 times according to the standard AB protocol. The products were puriﬁed on Centrisep spin-columns, vacuum- dried, resuspended in 2 ml Accutrac dye, heated to 90 C for 2 min and analysed in an AB 377 sequencer (25). Raw sequence runs were edited and assembled using ‘Autoassem- bler’, formatted with ‘Gene Jockey’, and analyzed using the web-based BLAST programs at NCBI (http://www.ncbi.nlm. nih.gov) (26) and REBASE (1). Purification of PstII PstII was puriﬁed from E.coli TOP10 cells transformed with one of the recombinant clones from the sequencing study (p4409). Typically, cells were grown in 1 l of LB medium containing ampicillin (50 mg/ml) at 37 C and 245 r.p.m. until an OD600 of 2.0. The harvested cells were resuspended in 20 ml of Buffer A [20 mM Tris–HCl (pH 8.0), 1 mM EDTA, 10% (v/v) glycerol, 100 mM NaCl, 0.5 mM DTT] and disrupted by sonication on ice. The cell extract was cla- riﬁed by low speed centrifugation at 23 000 g for 10 min followed by 100 000 g for 2 h. The supernatant was dialysed against Buffer B [50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 50 mM NaCl, 1 mM DTT] and the ﬁltered dialysate loaded onto two 20 ml Hi-prep heparin Sepharose FF columns (Amersham Biosciences) connected in series and equilibrated in Buffer B. Bound proteins were eluted with a linear gradient of NaCl in Buffer B (0.10–0.75 M, 420 ml). Fractions were analysed for DNA cleavage activity on pLJP11b and by SDS– PAGE. PstII was eluted in a peak at 0.41 M NaCl. Signiﬁcant loss of Res subunit was noted (Res eluted in a far broader peak than Mod). Those fractions containing approximately equal proportions of both Res and Mod were pooled and further puriﬁed on an 8 ml Mono-Q column (Amersham Biosciences) equilibrated with Buffer B. PstII was eluted with a linear gradient of NaCl in Buffer B (0.10–1.00 M, 200 ml). Fractions were analysed for DNA cleavage activity on pLJP11b and by SDS–PAGE. PstII eluted in a peak at 0.43 M NaCl with little separation of Res and Mod subunits. The fractions containing pure Res and Mod (>95% homogeneity) were pooled and concentrated by spin ﬁltration in Amicon Ultra-15 columns (30 000 MWCO, Millipore). The ﬁnal retentate was supple- mented to 50% (v/v) glycerol and stored at 20 C with no loss of activity upon storage noted. Protein concentration was determined from the UV absorption at 280 nm using an extinc- tion coefﬁcient derived from the aromatic amino acid composition of the predicted amino acid sequences. All PstII concentrations are quoted in terms of a mixture of Res and Mod subunits in the ratio 1:3 as estimated from densitometry of Coomassie-stained SDS gels and assuming a linear rela- tionship between protein size and dye intensity. DNA cleavage assays with PstII and/or EcoP15I Cleavage reactions contained 5 nM plasmid DNA or 500 ng phage DNA, and 129 nM PstII mixture (or 1/10 vol lysate/ column fraction), and/or 25 nM EcoP15I in NEB 4 [20 mM