Figure 2

CRP and Fis participate in the repression of rpoS . A) RT-PCR assay for mRNAs examining transcription of the rpoS gene from S. Typhi wild-type (STH2370), Δcrp, Δfis, Δcrp Δfis double mutant, and the complemented strains Δcrp/pTCRP and Δfis/pTFIS. 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 (control) or LB + 0.4% glucose, 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. *p < 0.05; **p < 0.001 (ANOVA). B) Immunodetection of epitope-tagged RpoS from S. Typhi hlyE-3xFLAG (par.: parental strain), S. Typhi rpoS-3xFLAG Δcrp, and S. Typhi hlyE-3xFLAG Δfis. Bacteria 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 or LB + 0.4% glucose, and incubated 1 h at 37°C. After the incubation, total proteins were obtained, and 20 μg were used to detect RpoS accumulation as described in Methods. C) In silico sequence analysis of CRP and Fis binding boxes in S. Typhi CT18 rpoS promoter. The S. Typhi rpoS promoter contains conserved binding boxes of S. Typhimurium rpoS promoter according to the previous evidence [19, 25]. Segmented underlined sequences indicate putative CRP boxes. Underlined sequences indicate putative Fis boxes. Asterisk shows a shared region of each box at the marked position. In bold, transcription start site, and short underlined sequences indicate predicted −35 and −10 promoter regions.