Bakermans, et al.

Supplemental Material

Materials and methods

Purification of restriction endonuclease and restriction assay

P. arcticus 273-4 was grown in 600 ml LB at room temperature. After 3 days, cells were collected by centrifugation at 6000×g for 10 min at 4°C, resuspended in 35 ml ice-cold Buffer A (10 mM Tris-HCl, 0.2 mM MgCl2, 0.2 mM EDTA, 2.0 mM ß-mercaptoethanol, pH 7.4) plus 0.4 mM PMSF, and sonicated 60-90 sec 5 to 10 times with 1 min incubations on ice between sonications. Cell debris was removed by centrifugation at 1000×g for 45 min at 4°C. The supernatant was transferred to a clean 50 ml centrifuge tube; NaCl and polyethyleneimine was added to a final concentration of 0.1 mM and 1% (v/v), respectively; and mixed gently. Precipitated nucleic acids were removed by centrifugation at 15,000×g for 10 min at 4°C. The supernatant was transferred to a 250 ml centrifuge bottle; 100% saturated (NH4)2SO4 was added to a final concentration of 70%; and mixed gently at 4°C for 0.5 to 4 h. Precipitated proteins were collected by centrifugation at 25,000×g for 30 min at 4°C; the supernatant was decanted and discarded, while proteins were resuspended in 10 ml Buffer B (20 mM KH2PO4, 0.2 mM MgCl2, 0.2 mM EDTA, 2.0 mM ß-mercaptoethanol, pH 7.4). The precipitated nucleic acid pellet was re-extracted with 10 ml of Buffer A plus 0.6 M NaCl. Nucleic acids were removed by centrifugation 25,000×g for 10 min at 4°C; the supernatant was saved. The resuspended protein pellet and the supernatant (from the previous step) were pooled, placed in 10 mm (MW cut off 6,000-8,000) Spectra/Por membrane dialysis tubing, and dialyzed overnight against 1 l Buffer B, twice. Dialyzed samples were loaded on a Whatman P11 phosphocellulose column (1.5×10 cm) previously equilibrated with Buffer B. The column was washed three times with Buffer B plus 10% glycerol. Proteins were eluted with a 0 to 1.2 M NaCl gradient in Buffer B plus 10% glycerol using a gradient maker and fraction collector set to 50 drops (~3 ml). Fractions were stored at 4°C; 4 μl of each fraction was assayed for restriction activity as below. Fractions with significant restriction activity were pooled, dialyzed against 50× excess Buffer B plus 50% glycerol, and stored in glycerol at 20°C.All column chromatography was performed at 4°C.

The DNA was a 2 Kb fragment (of the P. arcticus 273-4 genome containing the DpnC gene) that was isolated from vectors that were grown in E.coli DH5α or E. coli GM2163. DNA (0.5 μg) was digested with 2 l partially purified P. arcticus 273-4 enzyme or 2 l DpnI in 1X New England Biolabs Buffer 3 (final volume of 20 μl) at 37°C for 1.5 h.

Reverse-transcriptase-PCR analysis of expression of dctTUF operon

Strains were grown in MM as above at 22 or 4°C; when the optical density at 600 nm reached an average of 0.200 RNA was extracted using a Qiagen RNeasy Midi Kit and stored at -80°C. RNA was digested with Promega DNase I according to the manufacturer’s instructions. cDNA was synthesized from RNA using Invitrogen Superscript III Reverse Transcriptase according to the manufacturer’s instructions. PCR was performed as above at an annealing temperature of 58C with the following primer pairs: dctT-F (CAAAACCTTTGGCGTCAACT) and dctT-R (TCCGAGCATTCACTTCTGTG); dctU-F (GAGTTGATGGCGTTGAGGAT) and dctU-R (TTCGCGAAGATACTGCTCCT); and dctF-F (GCTAGCTTCGATCCTCTTGG) and dctF-R (CCCAATCACACCAATGACAG). PCR products were visualized by agarose gel electrophoresis and staining with ethidium bromide.

Supplemental Fig. 1 Restriction of methylated vs. nonmethylated DNA by P. arcticus 273-4restriction enzyme. Enzyme is D = Dpn I or P = partially purified enzyme from P. arcticus 273-4. Marker is 1 Kb ladder (Promega).

Supplemental Fig. 2 Replication of plasmids in P. arcticus 273-4. A. XbaI digest of pRL1062a (~13 Kb) isolated from E.coli MV1190 (3 lanes) and P. arcticus 273-4 (3 lanes). B. HindIII digest of pRL412 (9.5 Kb) isolated from E. coli GM2163 (1 lane) and P. arcticus 273-4 (4 lanes). M = marker, 1 Kbp ladder.

Supplemental Fig. 3 RT-PCR analysis of dctTUF transcripts from RNA extracted from actively growing cultures of P. arcticus 273-4. Expected size of PCR products is noted. M = 100 bp ladder, + = positive control (genomic DNA), - = negative control (reagents only), RT = Reverse transcriptase, w = wildtype, Δ = mutant.

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