Fig 1.
β-galactosidase activity exhibited by different transposon mutants and trans-complemented strains.
The data represent mean values (± standard deviation) from three independent experiments performed in triplicate. Asterisks indicate statistically significant differences (**—p<0.01; ***—p<0.001) according to Student’s unpaired t-test.
Table 1.
Characterization of the sites of Tn5-B22 transposition in Y. enterocolitica mutants.
Table 2.
Putative OmpR-regulated proteins of Y. enterocolitica Ye9 identified by SDS-PAGE and LC-MS/MSa.
Fig 2.
Influence of OmpR activity on the Y. enterocolitica membrane protein profile.
Proteins were isolated from strains Ye9 (wild type) and AR4 (ompR mutant) grown overnight in LB medium at 27°C or 37°C. In each case, 50 μg of protein were separated by SDS-PAGE and visualized by Coomassie blue staining. Putative OmpR-regulated proteins subsequently identified by LC-MS/MS are indicated by arrows. The bands were named according to their migration in the 12% polyacrylamide gel relative to the molecular weight standards.
Fig 3.
RT-PCR analysis of the acrRdsrE operon.
The analysis was performed using total RNA isolated from strain Ye9 grown in LB medium at 27°C. The size of the amplified fragment was estimated by comparison with the size marker DNAs (lane M). A negative control reaction was performed using DNase-treated RNA as the template.
Fig 4.
Intergenic sequences upstream of the Y. enterocolitica acrR and acrAB operons.
The initiation codons (ATG) are in bold, and putative -35 and -10 promoter elements are underlined. The putative OmpR binding sites identified by in silico analysis (R1, R2) are shaded gray. Potential binding sites for AcrR (A1, A2) are boxed. Potential binding site elements aligned with the E. coli consensus sequences, with the percentage identities presented in parentheses.
Fig 5.
Effect of OmpR and growth phase on acrR and acrAB promoter activity.
Strains Ye9 and AR4 carrying the promoter-gfp fusion constructs were cultivated in LB medium at 27°C to exponential phase (A) and stationary phase (B). Data represent mean fluorescence activity values normalized to the OD600 of the culture (± standard deviation) from three independent experiments performed in triplicate. The significance of differences between the values was calculated using Student’s unpaired t-test (ns [non significant]—p>0.05, *—p<0.05, **—p<0.01).
Fig 6.
The influence of growth temperature on acrR and acrAB promoter activity.
Strains Ye9 and AR4 carrying the promoter-gfp fusion constructs were cultivated in LB medium at 27°C, 30°C or 37°C to exponential phase. Data represent mean fluorescence activity values normalized to the OD600 of the culture (± standard deviation) from three independent experiments performed in triplicate. Significance of differences between the values was calculated using Student’s unpaired t-test (**—p<0.01, ***—p<0.001).
Table 3.
Modulation of acrR and acrAB expression in strains Ye9N and AR4 by different stressors.
Fig 7.
Interaction of purified OmpR with the acrR and acrAB promoter regions examined by electrophoretic mobility shift assays.
EMSAs using Y. enterocolitica acrR (upper panel) or acrAB (lower panel) promoter fragments with in vitro phosphorylated OmpR protein (lane 1—no protein; lane 2–1.2 μM; lane 3–2.5 μM; lane 4–3.7 μM; lane 5–5 μM; lane 6–6.2 μM; lane 7–7.5 μM). A 16S rDNA fragment was included as a control in all reactions to confirm specific binding.
Table 4.
Strains and plasmids used in this study.