BfmR regulates the expression of rhlR in a direct manner.

A) EMSA shows that 6His-BfmR directly binds to the promoter DNA of rhlR but not to that of rhlC. B) Electropherograms show the protection pattern of the rhlR promoter after digestion with DNase I following incubation in the absence or the presence of 6His-BfmR. There are three BfmR-protected regions (I, II, and III) in the promoter of the rhlR. BfmR-protected regions I harbors a putative BfmR-binding motif, which was highlighted in bold and in italics, as indicated. C) The relative expression of rhlR-lux in the wild-type MPAO1 (harboring PAK1900), the ΔbfmS strain (harboring PAK1900), the ΔbfmS/p-bfmS strain, the ΔbfmRS strain (harboring PAK1900), and the ΔbfmRS/p-bfmR strain. The relative gene expression in the wild-type MPAO1 was set to 1, and the other values were adjusted accordingly. D) The expression of rhlR-lux and rhlR-D-lux in the wild-type MPAO1, the ΔbfmS strain and the ΔbfmRS strain, as indicated. Bacteria were grown in low phosphate (0.32 mM) M8-glutamate minimal medium supplemented with 2% glucose at 37°C for 48 h. Values represent means ± SEM. The assays were independently repeated at least three times and the data shown represent comparable results.