Effect of DK2 lineage-specific amino acid substitutions in BfmS on the activation of BfmR.

In all panels, MPAO1 and ΔbfmS harbor plasmid PAK1900, respectively. A) The relative expression of bfmR-lux in the wild-type MPAO1 and its derivatives when bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm), as indicated. Values are relative to MPAO1 (set to 1). B) Relative amount of C4-HSL measured by the pDO100 (pKD-rhlA) system. MPAO1 and its derivatives were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm). Supernatants were subsequently prepared and measured for the relative C4-HSL contents. C) The relative expression of bfmR-lux in the wild-type MPAO1 and its derivatives when bacteria were grown in M8-glutamate minimal medium supplemented with 0.082% sodium acetate at 37°C for 36 h with shaking (250 rpm). Values are relative to MPAO1 (set to 1). D) The bfmR-lux activity in ΔbfmS/p-bfmS strain (bfmS), ΔbfmS/p-bfmSH238A strain (H238A), ΔbfmS/p-bfmSL181P/E376Q (L181P/E376Q), and ΔbfmS/p-bfmSL181P/E376Q/H238A strain (L181P/E376Q/H238A) when bacteria were grown in M8-glutamate minimal medium supplemented with 0.2% glucose at 37°C for 36 h with shaking (250 rpm). Values are relative to ΔbfmS/p-bfmS strain (set to 1). In A) to D), results are representative of three independent experiments and values represent means ± SEM. E) Phos-tag analysis shows that amino acid substitution L181P/E376Q, but not the L181P/E376Q/H238A, causes overproduction of phosphorylated and unphosphorylated BfmR. Western blot analysis of BfmS showing that missense mutations in bfmS do not affect the protein levels of BfmS, and immunoblots for RNAP (RNA polymerase) served as loading control. Cell lysates of ΔbfmRS::BfmR-Flag/p-bfmS strain (bfmS), ΔbfmRS::BfmR-Flag/p-bfmSL181P/E376Q (L181P/E376Q), and ΔbfmRS::BfmR-Flag/p-bfmSL181P/E376Q/H238A strain (L181P/E376Q/H238A) were used as described in Materials and Methods section.