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.</p

The <i>luxS</i> gene is necessary for normal expression of SPI-1 and virulence.

<p>(A) Transcriptional levels of the SPI-1 genes <i>sicA, sopB,</i> and <i>sopE</i> were determined by qRT-PCR. Overnight cultures of wild-type (WT) and mutant strains were diluted in fresh LB and mRNA samples were prepared from stationary phase of static cultures. Values are means and standard deviations of three independent experiments. (B and C) Six-week-old BALB/c mice (n = 10) were infected orally with 10⁷ CFU (B) or intraperitoneally (I.P.) with 10³ CFU (C) <i>Salmonella</i> strains. Mice surviving after infection were monitored daily for two weeks.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

The overexpression of LsrR decreased <i>Salmonella</i> invasion into HEp-2 epithelial cells, even when the requirement for motility is bypassed through centrifugation.

<p>Monolayers of HEp-2 epithelial cells were infected with the wild-type (WT) <i>Salmonella</i>, WT harboring backbone plasmid, pUHE21-2<i>lacI^q</i>, and WT harboring pJH1 strains in the presence or absence of 100 µM IPTG. To exclude the requirement of motility, mild centrifugation was employed (centri.). The numbers of internalized bacteria were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037059#s4" target="_blank">Methods</a>. Values represent the relative amount of internalized bacteria and have been normalized to the level of internalization of WT strain, which was set at 1.00. Values are the average and standard deviation from three independent experiments, each done in triplicate.</p

LsrR negatively controls the expression of flagella.

<p>(A) Wild-type (WT) and mutant strains carrying <i>fliC-lacZ</i> fusion on chromosome were diluted in LB medium grown with shaking, and β-galactosidase activity (Miller units) was determined at 4 h. Values are the means and standard deviation of three independent experiments. (B, left) Wild-type (WT) <i>Salmonella</i> carrying a chromosomal <i>invF-lacZ</i> fusion and either the control plasmid, pUHE21-2<i>lacI^q</i> or the <i>lasR</i>⁺ plasmid pJH1 were grown in LB with shaking for 4 h. Production of LsrR was induced by addition of 100 µM IPTG. (B, right) WT strain carrying pJH1 was grown in LB or LB with 100 µM IPTG, with shaking for 4 h. The mRNA levels of flagella genes were determined by qRT-PCR. Values are means and standard deviations of three independent experiments. (C) Representative SDS-PAGE gel of secreted proteins. Overnight cultures of the WT strain harboring either pUHE21-2<i>lacI^q</i> or pJH1 were diluted (1∶100) into fresh LB broth in the presence or absence of 100 µM IPTG and grown for 4 h with (aerobic) or without (anaerobic) shaking. Secreted proteins were recovered from cell-free spent culture media by TCA precipitation. (D) Electron microscopic observation of flagella using negative stain. Samples were prepared from WT cells harboring either pUHE21-2<i>lacI^q</i> or pJH1 grown in LB with 100 µM IPTG. The scale bar indicates 0.5 µm. (E) Phenotypic assay for motility was performed to confirm the down-regulation of flagella genes in LsrR-overexpressing <i>Salmonella</i> cells. A 1 µl aliquot of washed WT cells harboring either pUHE21-2<i>lacI^q</i> or pJH1 was stab inoculated into 0.3% LB agar with or without 100 µM IPTG. The images were taken following 8 h of growth at 37°C.</p

LsrR is involved in the regulation of SPI-1 expression and invasion of <i>S. typhimurium</i>.

<p>(A) Wild-type (WT) and mutant strains carrying a chromosomal <i>invF-lacZ</i> fusion were diluted in LB medium and β-galactosidase activity (Miller units) was determined at 4 h of cultures grown with shaking. Values are the means and standard deviation of three independent experiments. (B) Monolayers of HEp-2 epithelial cells were infected with the wild-type (WT) and mutant strains and numbers of internalized bacteria were determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037059#s4" target="_blank">Methods</a>). Values represent the relative amount of internalized bacteria normalized to the level of internalization of the WT strain, which was set to 1.00. Values are the average and standard deviation from three independent experiments, each done in triplicate.</p

LsrR negatively controls the expression of SPI-1 genes.

<p>(A) Wild-type (WT) <i>Salmonella</i> carrying a chromosomal <i>invF-lacZ</i> fusion and either the control plasmid, pUHE21-2<i>lacI^q</i> or the <i>lasR</i>⁺ plasmid pJH1 were grown in LB with shaking for 4 h. Production of LsrR was induced by the addition of 100 µM IPTG. (B) WT <i>Salmonella</i> carrying pJH1 were grown LB or LB supplemented with 100 µM IPTG, with shaking for 4 h. The mRNA levels of SPI-1 genes were determined by qRT-PCR. Shown in (A) and (B) are the mean values and standard deviation of three independent experiments. (C) Western blot analysis was conducted on cell extracts prepared from the strain harboring either pUHE21-2<i>lacI^q</i> or pJH1 grown in LB with 100 µM IPTG, with shaking for 4 h. These strains express the SopB protein tagged with a HA-epitope from the normal chromosomal location.</p