Fig 4. glmS1 suppresses ΔglmR by abolishing negative feedback regulation of glmS expression.

(A) Representative growth curves in MH medium (n>3) showing the effect of point mutation glmS1 in ΔglmR compared to WT, glmS1 and ΔglmR. (B) Disc diffusion assay showing the effect of glmS1 on CEF sensitivity phenotype of ΔglmR. 6 μg CEF was used in the assay. Standard deviation (error bars) is based on at least three biological replicates. Three asterisks indicate significant difference with P <0.001 using Tukey test. (C) qRT-PCR results showing glmS mRNA level in glmS1, ΔglmR and ΔglmR glmS1 relative to WT from cells harvested at OD₆₀₀ of 0.5 grown in LB. Standard deviation (error bars) is based on at least three biological replicates. Statistical significance is determined by Tukey test where three asterisks indicate P <0.001 and NS is non-significant (P >0.05). (D) Western blot analysis of GlmS protein in ΔglmR and ΔglmR glmS1 using anti-GlmS antibodies. 5 μg of total protein was loaded in each lane.