Fig 4. CRP-cAMP-mediated repression of HilD in mid-logarithmic growth requires the 3’UTR of hilD.

(A) hilD transcriptional expression was monitored using three different hilD-lacZ reporter fusions; these comprise either hilD to position +76 (within the hilD ORF), to position +965 (including the full-length hilD ORF), or to position +1,235 (including the hilD ORF and the hilD 3’UTR). Transcriptional studies were performed in the wild-type (WT) and Δcrp derivative strains grown in LB at 37°C up to an OD_600nm of 0.4. The transcriptional expression is shown in relative values. In each case the reference (WT) value was set to one. Miller units in WT strains, hilD76-lacZ 63.3 +/- 5.9; hilD965-lacZ 5193.0 +/- 334.6; hilD1235-lacZ 38.3+/- 2.0. (B) Immunodetection of HilD-3xFLAG. Two different genetic constructs were used: one containing the hilD 3’UTR (+UTR) and the other lacking that region (-UTR). Immunodetection was assessed in whole cell extracts from cultures of the WT and Δcrp derivative strains grown as in A. (C) Immunodetection of the SPI-1 encoded SipA-3xFLAG protein was performed on whole cell extracts from two independent cultures of the WT and Δcrp derivative strains, either in the presence (+UTR) or the absence (-UTR) of the hilD 3’UTR. Cultures were grown as in A. In B and C, Coomassie Blue staining of the whole cell extracts serve as loading controls. Full length images of the Western blots, including molecular mass markers, are shown in S12 Fig. (D) sipC transcriptional expression was monitored in cultures grown as in A of the WT and Δcrp derivative strains carrying a hilD native gene or a derivative hilD lacking the 3’UTR. The transcriptional expression is shown in relative values. In each background (+UTR, -UTR) the activity reference of WT was set to one. Miller units in WT strains, UTR⁺ 73.6 +/- 11.8; UTR^- 13783.0 +/- 1142.5. In A and D, the β-galactosidase activity was determined for three independent cultures, average and standard deviation is shown. **, p< 0.01; ***, p< 0.001.