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

<p>In all panels, MPAO1 and Δ<i>bfmS</i> harbor plasmid PAK1900, respectively. <b>A</b>) The relative expression of <i>bfmR-lux</i> 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>B</b>) Relative amount of C4-HSL measured by the pDO100 (pKD-<i>rhlA</i>) 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. <b>C</b>) The relative expression of <i>bfmR-lux</i> 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). <b>D</b>) The <i>bfmR-lux</i> activity in Δ<i>bfmS</i>/p-<i>bfmS</i> strain (<i>bfmS</i>), Δ<i>bfmS</i>/p-<i>bfmS_H238A</i> strain (<i>H238A</i>), Δ<i>bfmS</i>/p-<i>bfmS_L181P/E376Q</i> (<i>L181P/E376Q</i>), and Δ<i>bfmS</i>/p-<i>bfmS_{L181P/E376Q/H238A}</i> strain (<i>L181P/E376Q/H238A</i>) 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 Δ<i>bfmS</i>/p-<i>bfmS</i> strain (set to 1). In <b>A</b>) to <b>D</b>), results are representative of three independent experiments and values represent means ± SEM. <b>E</b>) 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 <i>bfmS</i> do not affect the protein levels of BfmS, and immunoblots for RNAP (RNA polymerase) served as loading control. Cell lysates of Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS</i> strain (<i>bfmS</i>), Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS_L181P/E376Q</i> (<i>L181P/E376Q</i>), and Δ<i>bfmRS</i>::<i>BfmR</i>-<i>Flag</i>/p-<i>bfmS_{L181P/E376Q/H238A}</i> strain (<i>L181P/E376Q/H238A</i>) were used as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004340#s4" target="_blank">Materials and Methods</a> section.