Effect of <i>crp</i> deletion on the expression of upstream activators of <i>hilA</i>.

<p>(A) Relative quantification of <i>hilD</i>, <i>hilC</i> and <i>rtsA</i> mRNA by qRT-PCR. Total RNA samples were extracted from cultures of the wild-type (WT) and Δ<i>crp</i> derivative strains. Results are expressed as fold changes between WT and Δ<i>crp</i>. Detection of <i>gapA</i> (GAPDH) was used as an internal control (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007401#sec011" target="_blank">Material and Methods</a>). (B) <i>hilA</i> transcriptional expression (β-galactosidase activity) was monitored in <i>crp</i>⁺ and Δ<i>crp</i> strains in different genetic backgrounds: WT, Δ<i>hilC</i>, Δ<i>hilD</i> and Δ<i>rtsA</i>. (C) <i>hilC</i> and <i>hilD</i> transcriptional expression (β-galactosidase activity) was monitored. For <i>hilC</i>, WT, Δ<i>crp</i> and Δ<i>crp</i> Δ<i>hilD</i> derivative strains carrying a <i>hilC</i>-<i>lacZ</i>, chromosomal fusion were used. For <i>hilD</i>, WT, Δ<i>crp</i> and Δ<i>crp</i> Δ<i>hilC</i> carrying the <i>hilD</i>1235-<i>lacZ</i> (<i>hilD</i>⁺, 3’UTR⁺) were used. In A, B and C, data correspond to the average and standard deviation of three independent experiments. **, <i>p</i>< 0.01; ***, <i>p</i>< 0.001. (D) Northern blot analysis for <i>hilD</i> mRNA. Total RNA samples were extracted from cultures of the WT and Δ<i>crp</i> strains. tmRNA was detected as loading control. For quantification, the ratio [<i>hilD</i> mRNA/tmRNA] was calculated. Full length image of the northern blot is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007401#pgen.1007401.s012" target="_blank">S12 Fig</a>. In all cases cultures were grown in LB at 37°C up to an OD_600nm of 0.4.