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Bakermans, et al.
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Supplemental Material
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Materials and methods
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Purification of restriction endonuclease and restriction assay
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P. arcticus 273-4 was grown in 600 ml LB at room temperature. After 3 days, cells were
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collected by centrifugation at 6000×g for 10 min at 4°C, resuspended in 35 ml ice-cold Buffer
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A (10 mM Tris-HCl, 0.2 mM MgCl2, 0.2 mM EDTA, 2.0 mM ß-mercaptoethanol, pH 7.4)
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plus 0.4 mM PMSF, and sonicated 60-90 sec 5 to 10 times with 1 min incubations on ice
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between sonications. Cell debris was removed by centrifugation at 1000×g for 45 min at 4°C.
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The supernatant was transferred to a clean 50 ml centrifuge tube; NaCl and
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polyethyleneimine was added to a final concentration of 0.1 mM and 1% (v/v), respectively;
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and mixed gently. Precipitated nucleic acids were removed by centrifugation at 15,000×g for
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10 min at 4°C. The supernatant was transferred to a 250 ml centrifuge bottle; 100% saturated
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(NH4)2SO4 was added to a final concentration of 70%; and mixed gently at 4°C for 0.5 to 4 h.
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Precipitated proteins were collected by centrifugation at 25,000×g for 30 min at 4°C; the
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supernatant was decanted and discarded, while proteins were resuspended in 10 ml Buffer B
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(20 mM KH2PO4, 0.2 mM MgCl2, 0.2 mM EDTA, 2.0 mM ß-mercaptoethanol, pH 7.4). The
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precipitated nucleic acid pellet was re-extracted with 10 ml of Buffer A plus 0.6 M NaCl.
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Nucleic acids were removed by centrifugation 25,000×g for 10 min at 4°C; the supernatant
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was saved. The resuspended protein pellet and the supernatant (from the previous step) were
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pooled, placed in 10 mm (MW cut off 6,000-8,000) Spectra/Por membrane dialysis tubing,
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and dialyzed overnight against 1 l Buffer B, twice. Dialyzed samples were loaded on a
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Whatman P11 phosphocellulose column (1.5×10 cm) previously equilibrated with Buffer B.
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The column was washed three times with Buffer B plus 10% glycerol. Proteins were eluted
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with a 0 to 1.2 M NaCl gradient in Buffer B plus 10% glycerol using a gradient maker and
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fraction collector set to 50 drops (~3 ml). Fractions were stored at 4°C; 4 μl of each fraction
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was assayed for restriction activity as below. Fractions with significant restriction activity
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were pooled, dialyzed against 50× excess Buffer B plus 50% glycerol, and stored in glycerol
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at -20°C. All column chromatography was performed at 4°C.
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The DNA was a 2 Kb fragment (of the P. arcticus 273-4 genome containing the DpnC
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gene) that was isolated from vectors that were grown in E.coli DH5α or E. coli GM2163.
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DNA (0.5 μg) was digested with 2 l partially purified P. arcticus 273-4 enzyme or 2 l DpnI
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in 1X New England Biolabs Buffer 3 (final volume of 20 μl) at 37°C for 1.5 h.
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Reverse-transcriptase-PCR analysis of expression of dctTUF operon
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Strains were grown in MM as above at 22 or 4°C; when the optical density at 600 nm reached
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an average of 0.200 RNA was extracted using a Qiagen RNeasy Midi Kit and stored at -80°C.
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RNA was digested with Promega DNase I according to the manufacturer’s instructions.
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cDNA was synthesized from RNA using Invitrogen Superscript III Reverse Transcriptase
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according to the manufacturer’s instructions. PCR was performed as above at an annealing
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temperature of 58C with the following primer pairs: dctT-F
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(CAAAACCTTTGGCGTCAACT) and dctT-R (TCCGAGCATTCACTTCTGTG); dctU-F
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(GAGTTGATGGCGTTGAGGAT) and dctU-R (TTCGCGAAGATACTGCTCCT); and
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dctF-F (GCTAGCTTCGATCCTCTTGG) and dctF-R (CCCAATCACACCAATGACAG).
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PCR products were visualized by agarose gel electrophoresis and staining with ethidium
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bromide.
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Methylated +
Enzyme
-
+
D
+
P
D
P
Size
(bp)
2013
1543
470
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Supplemental Fig. 1 Restriction of methylated vs. nonmethylated DNA by P. arcticus 273-4
restriction enzyme. Enzyme is D = Dpn I or P = partially purified enzyme from P. arcticus
273-4. Marker is 1 Kb ladder (Promega).
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2
A
B
M E.coli | P arc M E | P arc
arc
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arc
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.
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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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