Figure 3

PhoPQ two-component regulatory system participates in the hlyE induction via rpoS. A) RT-PCR assay for mRNAs examining transcription of the hlyE gene from S. Typhi wild-type (STH2370), ΔrpoS, ΔphoPQ, and the ΔrpoS ΔphoPQ double mutant, and the complemented strains ΔrpoS/pBRPOS and ΔphoPQ/pBPHOPQ. All the strains were grown in LB buffered at pH 7.0 to an OD₆₀₀ = 0.35, washed three times with PBS prior to be resuspended with LB buffered at pH 7.0 (control) or LB buffered at pH 5.0, and incubated 1 h at 37°C. After the incubation, total RNA were isolated for each strain. The same amount of RNA was applied for RT-PCR. The transcription levels were normalized against the 16 s transcript in all cases. The data correspond to mean values of three independent experiments performed in triplicate. Error bars correspond to the SD. B) RT-PCR assay for mRNAs examining transcription of the hlyE gene from S. Typhi wild-type (STH2370), ΔrpoS, ΔphoPQ, and the ΔrpoS ΔphoPQ double mutant. All the strains were grown in LB buffered at pH 7.0 to an OD₆₀₀ = 0.35, washed three times with PBS prior to be resuspended with M9 10 mM Mg²⁺(control) or M9 10 μM Mg²⁺, and incubated 1 h at 37°C. After the incubation, total RNA were isolated for each strain. The same amount of RNA was applied for RT-PCR. The transcription levels were normalized against the 16 s transcript in all cases. The data correspond to mean values of three independent experiments performed in triplicate. Error bars correspond to the SD. In all the cases, the numbers above the bars represent the fold of induction. *p < 0.05 (ANOVA).