Methylation Assays of Bacterial Cultures

Methylation assays were performed as previously described (Watts et al., 2011 (link)). Briefly, cultures were induced with 200 μM IPTG, centrifuged, washed and resuspended in chemotaxis buffer [0.1 mM K⁺EDTA, 10 mM KPO₄ pH 7.4, 10 mM Na-lactate, 1 mM MgSO₄, and 1 mM (NH₄)₂SO₄]. Then, 200 μg ml⁻¹ chloramphenicol was added to inhibit protein synthesis, and methylation was initiated by adding 9.7 μCi ml⁻¹ L-(methyl-³H) methionine (PerkinElmer, Waltham, MA). Reactions were stopped with 2 μl formaldehyde (per 1.02 ml reaction). After SDS-PAGE, gels were soaked for 30 min in Fluorohance™ (Research Products International, Mount Prospect, IL), then dried and exposed to autoradiography film at −80 °C for 2–4 days. Bands were quantified in the linear range using VisionWorks®LS Analysis Software (Analytik Jena, Upland, CA). Band densities were normalized by dividing by the concentration of protein in the formaldehyde-treated samples as determined in a BCA™ Protein Assay (Thermo Scientific). Statistical analyses were carried out using a two-tailed Student’s t-test. A value of P <0.05 was considered statistically significant.