Figure 1.
Cytosine deamination by M.SssI in vitro.
pUP41 (ApR, KnS) plasmid DNA was incubated with or without wild-type M.SssI (2-fold molar excess relative to CG sites) at 30°C for 4 h, and frequency of C-to-U deamination was determined by scoring the numbers of KnR and ApR transformants in E. coli ER2357 ung strain. SAM (160 µM) and 5’-amino-5’-deoxyadenosine (AA, 250 μM) were added to samples as indicated. Error bars represent standard error of the mean of at least three independent experiments (p<0.01).
Figure 2.
DNA methyltransferase activity of the F17S and G19D M.SssI mutants.
(A) Amino acid sequence alignment between segments of M.HpaII and M.SssI. Conserved residues of the FXGXG motif are in bold. The F17S and G19D substitutions are indicated below the sequence.
(B) Hin6I digestion of plasmids pBHNS-MSssI, pBHNS-MSssI(F17S) and pBHNS-MSssI(G19D) encoding WT or mutant M.SssI variants, respectively as indicated above the lanes. Plasmids were isolated from cultures grown for 4-6-8 h in the presence of arabinose to induce M.SssI expression. Resistance to Hin6I (recognition sequence GCGC) indicates M.SssI-specific methylation (R. Kazlauskiene, cited in REBASE [45]. Lane Unmeth., unmethylated pBHNS-MSssI(G19D) isolated from cells grown in the presence of glucose; Lane Undig., undigested pBHNS-MSssI; M, molecular weight marker (GeneRuler 1 kb DNA Ladder, Fermentas).
(C) Effect of WT and mutant M.SssI production on growth of E. coli mcrBC and mcrBC+ hosts. DH10B mcrBC contained pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) as indicated. DH5α mcrBC+ contained pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D). Bacteria were grown in LB/Ap medium at 30°C. MTase expression was induced at time 0 by arabinose. Error bars represent standard error of the mean of three independent experiments.
Figure 3.
Estimation of DNA MTase activity of the F17S and G19D M.SssI mutants by restriction enzyme protection assay.
Lambda phage DNA was incubated with different concentrations of WT and mutant M.SssI in the presence of SAM as described in Materials and Methods. Methylation status of the DNA was subsequently tested by digestion with the methylation sensitive restriction enzyme Hin6I, and the digestion was analyzed by agarose gel electrophoresis. Lane Undig., undigested plasmid; M, molecular weight marker (GeneRuler 1 kb Plus and GeneRuler 1 kb DNA Ladders, Fermentas).
M.SssI-mediated cytosine deamination in vivo was initially investigated using a two-plasmid-system, with the E. coli host containing the indicator plasmid pUP41 and one of the M.SssI-expressing plasmids pSTdC-MSssI, pSTdC-MSssI(F17S) or pSTdC-MSssI(G19D). The latter plasmids have pSC101 replicon and are compatible with pUP41. We observed elevated reversion frequency to kanamycin resistance with the Ung- host ER2357 expressing the G19D variant (not shown).
Figure 4.
Cytosine deamination by WT and mutant (F17S and G19D) M.SssI in vivo.
(A) E. coli ER2357-kanS ung and DH10B-kanS ung+ harbouring pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) encoding WT or mutant M.SssI were grown in the presence of arabinose to induce M.SssI expression. Frequency of KnR revertants was determined after 4 h growth. Due to poor growth after arabinose induction the reversion frequency of DH10B-kanS expressing the WT MT could not be reliably determined (Figure 4A). Empty bars, ung-; filled bars, ung+. Error bars represent standard error of the mean of three independent experiments (p<0.01).
(B) E. coli ER2357 ung and DH10B ung+ cells contained pUP41 (ApR KnS) and a compatible plasmid (pSTdC-MSssI, pSTdC-MSssI(F17S) or pSTdC-MSssI(G19D) expressing WT or mutant M.SssI. Mutation rates were calculated from reversion to KnR phenotype by fluctuation tests as described in Materials and Methods. Empty bars, ung-; filled bars, ung+. Results of the following number of experiments: no MTase (3), WT and F17S (2), G19D (4). Error bars represent standard error of the mean.
Figure 5.
Effect of sinefungin and 5’-amino-5’-deoxyadenosine on cytosine deamination activity of M.SssI and its mutants.
Plasmid pUP41 was incubated with purified M.SssI, M.SssI(F17S) or M.SssI(G19D) and frequency of KnR revertants was determined in ER2357 ung strain as described in Materials and Methods and legend of Figure 1. Sinefungin (SF) was used at 500 and 5’-amino-5’-deoxyadenosine (AA) at 250 µM concentration in samples indicated below the bars. Reversion frequency was derived from the ratio of the KnR and ApR transformants. Striped bar, no enzyme; empty bars, wild-type M.SssI; grey bars, M.SssI(F17S); black bars, M.SssI(G19D). Error bars represent standard error of the mean of five independent experiments (p<0.01).
Figure 6.
Comparison of cytosine and 5-methylcytosine deamination by M.SssI.
Unmethylated and in vivo M.SssI-specifically methylated pUP41 plasmid DNA was incubated with or without M.SssI(WT) and 5’-amino-5’-deoxyadenosine as indicated using conditions described in the legend of Figure 1. Frequency of reversion to KnR phenotype was determined for unmethylated pUP41 in ER2357 ung (empty bars), whereas reversion frequency of methylated pUP41 was determined in DH10B ung+ host (filled bars). Error bars represent standard error of the mean of at least three independent experiments (p<0.01).