DNA methyltransferase activity of the F17S and G19D M.SssI mutants.

<div><p>(<b>A</b>) 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. </p> <p>(<b>B</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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079003#B45" target="_blank">45</a>]. 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). </p> <p>(<b>C</b>) Effect of WT and mutant M.SssI production on growth of <i>E. coli </i><i>mcrBC</i> and <i>mcrBC</i>⁺ hosts. DH10B <i>mcrBC</i> contained pBHNS-MSssI, pBHNS-MSssI(F17S) or pBHNS-MSssI(G19D) as indicated. DH5α <i>mcrBC</i>^<i>+</i> 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. </p>