The regulatory function of LsrR was restored by exogenous autoinducer-2.

<p>(A) Schematic of the genomic context of the part of <i>lsr</i> operon, and the <i>lsrR</i>, and <i>lsrK</i> loci in the wild-type (WT) and P<i>cat-lsr</i> strains. The promoters of the <i>lsr</i> operon (<i>lsrA</i>) and <i>lsrK</i> have been replaced with the constitutively expressed promoter of the chloramphenicol resistant gene (P<i>cat</i>). (B and C) The P<i>cat</i>-<i>lsr</i> strains harboring pJH1 carrying a chromosomal <i>invF-lacZ</i> (B) or <i>fliC-lacZ</i> (C) transcriptional fusion were grown in LB containing 100 µM IPTG to induce LsrR expression, with shaking. When indicated, the signal molecule, AI-2 (DPD), was added at final concentrations of 48 and 144 µM. (D) The SDS-PAGE gel pattern of secreted flagella proteins was evaluated in the absence or presence of 100 µM IPTG and/or 144 µM DPD. The secreted proteins were recovered from cell-free spent culture media by TCA precipitation. (E) Western blot analysis was conducted on cell extracts prepared from P<i>cat</i>-<i>lsr</i> strains harboring pJH1 grown in LB or LB containing 100 µM IPTG and/or 144 µM DPD, with shaking. These strains express the SopB protein tagged with a HA-epitope (SopB-HA) from the normal chromosomal location. (F) Monolayer of HEp-2 epithelial cells were infected with wild-type (WT) <i>Salmonella</i>, WT <i>Salmonella</i> harboring pJH1, and P<i>cat</i>-<i>lsr Salmonella</i> harboring pJH1 in the presence or absence of 100 µM IPTG and/or 144 µM DPD. After the gentamicin treatment, the numbers of internalized bacteria were determined by plating the bacteria on LB agar following appropriate dilutions. Values represent the relative amount of the intracellular bacteria and have been standardized to the level of internalization of WT strain, which was set at 1.00. The values are the average and standard deviation from three independent experiments, each done in triplicate.