RP HPLC analysis of enterobactin release from the wild-type strain and the Δ<i>tolC</i> mutant.

<p>(A) Chromatograms of enterobactin prepared from the supernatants of the wild-type strain and the Δ<i>tolC</i> mutant. Peaks were identified using HPLC-grade standards. (B) The amount of enterobactin exported by Δ<i>tolC</i> mutant relative to that exported by the wild-type was calculated using the peak areas. The peak area corresponding to enterobactin released from the wild-type strain was defined as 100%.</p

Complementation of enterobactin release from the Δ<i>acrB acrD mdtABC</i> mutant using plasmids carrying <i>acrB</i>, <i>acrD</i>, or <i>mdtABC</i> genes.

<p>The amounts of enterobactin exported by deletion mutants, calculated using peak areas, are shown. The amount of enterobactin released from the wild-type strain harboring an empty vector was defined as 100%. The data corresponds to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (<i>p</i><0.01) determined using the two-tailed Student’s <i>t</i>-tests.</p

RP HPLC analysis of enterobactin released from deletion mutants of the RND-type efflux system genes.

<p>The amounts of enterobactin exported by deletion mutants calculated using each peak area are shown. The peak area corresponding to enterobactin released from the wild-type strain was defined as 100%. The data corresponds to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (<i>p</i><0.01) according to two-tailed Student’s <i>t</i>-tests.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Requirement of AcrB, AcrD, and MdtABC drug efflux systems for enterobactin export.

<p>Enterobactin was prepared from the supernatants of cultures of each multiple RND transporter mutant and analyzed by RP HPLC. The amount of enterobactin exported by each strain, calculated using each peak area, is shown. The amount of enterobactin released from the wild-type strain was defined as 100%. The data correspond to mean values from three independent replicates. The bars indicate standard deviations. Asterisks indicate statistically significant differences (<i>p</i><0.01) according to the two-tailed Student’s <i>t</i>-tests.</p

Proposed model of enterobactin export in <i>E. coli.</i>

<p>Enterobactin is synthesized in the cytoplasm and exported to the periplasm by EntS. The RND transporters AcrB, AcrD, and MdtABC capture enterobactin in the periplasm and then export it to the growth medium throughout the outer membrane channel TolC.</p

Data_Sheet_1_Major primary bile salts repress Salmonella enterica serovar Typhimurium invasiveness partly via the efflux regulatory locus ramRA.PDF

Bile represses Salmonella enterica serovar Typhimurium (S. Typhimurium) intestinal cell invasion, but it remains unclear which bile components and mechanisms are implicated. Previous studies reported that bile inhibits the RamR binding to the ramA promoter, resulting in ramA increased transcription, and that ramA overexpression is associated to decreased expression of type III secretion system 1 (TTSS-1) invasion genes and to impaired intestinal cell invasiveness in S. Typhimurium. In this study, we assessed the possible involvement of the ramRA multidrug efflux regulatory locus and individual bile salts in the bile-mediated repression of S. Typhimurium invasion, using Caco-2 intestinal epithelial cells and S. Typhimurium strain ATCC 14028s. Our results indicate that (i) major primary bile salts, chenodeoxycholate and its conjugated-derivative salts, cholate, and deoxycholate, activate ramA transcription in a RamR-dependent manner, and (ii) it results in repression of hilA, encoding the master activator of TTSS-1 genes, and as a consequence in the repression of cellular invasiveness. On the other hand, crude ox bile extract and cholate were also shown to repress the transcription of hilA independently of RamR, and to inhibit cell invasion independently of ramRA. Altogether, these data suggest that bile-mediated repression of S. Typhimurium invasion occurs through pleiotropic effects involving partly ramRA, as well as other unknown regulatory pathways. Bile components other than the bile salts used in this study might also participate in this phenomenon.</p

Effects of D13-9001 on FDG degradation determined by the microfluidic channel device.

<p>(A) Bright-field (top) and fluorescence images (bottom) of the <i>E. coli</i> wild-type, ΔB, and ΔC cells treated with different concentrations of PP. (B) Distributions of the fluorescence intensities of the cells and channels measured in the image shown in (A).</p

Effects of PAβN on FDG degradation determined by the microfluidic channel device.

<p>(A) Bright-field (top) and fluorescence images (bottom) of the <i>E. coli</i> wild-type, ΔB, and ΔC cells treated with different concentrations of PAβN. (B) Distributions of the fluorescence intensities of the cells and channels measured in the image shown in (A).</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p