Figure 5. Phosphosite mutation of MpGSMT affects methyltransferase activity and growth rate at elevated osmolarity.

(a) The relative GMT and SMT activities of recombinant WT MpGSMT and various mutant forms. The methyltransferase activities were performed by modified acid-washed charcoal method under 1.0 M of KCl with 0.5 M glycine or sarcosine as the substrate. All the data points were averaged by triplicate experiments and displayed as percentages relative to wild-type MpGSMT. The effect of salt adaptation was assayed by the growth of E. coli MKH13 containing WT or mutant (T68A and T68D) MpGSMT co-expressing with SDMT in M9 minimal media supplemented with (b) 0 mM, (c) 500 mM, or (d) 700 mM NaCl. Growth rate was monitored by measuring 600 nm optical densiometry (OD₆₀₀) over 24 h at 37 °C. E. coli strain MKH13 without a recombinant GSMT²¹ had the same growth rate as the T68D mutant. The effect of salt shock shown in the inset of (b,c) was measured by drop tests of serious dilution from 10⁰ to 10⁻⁷ of overnight cultures on agar plates, but cells failed to grow on agar plate with (d) 700 mM NaCl. Insect in (d) indicates the growth curve of T68D mutant with a large time-scale over 48 hours.