CsrA modulates the expression of a thiamine pyrophosphate (TPP) riboswitch element.
A) Schematic representation of the thi-operon in L. pneumophila including the transcriptional start site (TSS), the CsrA-binding region, the thi element of the predicted TPP riboswitch and a predicted transcription termination site upstream of the start codon (AUG). The CsrA-binding site is overlapping the thi element of the TPP riboswitch. This organization suggests that CsrA is implicated in the fine-tuning of the expression of the downstream thi-operon most probably due to conformational changes in the secondary RNA structure. B) EMSA with 200nM of biotinylated thi-element (TPP) RNA and purified CsrA: Lane 1: no CsrA, lane 2: 1.0 μM CsrA, lane 3: 2.0 μM CsrA, lane 4: 5.0 μM CsrA, lane 5: 5.0 μM CsrA + 2.0 μM unlabeled RsmZ. C) Beta-lactamase (BlaM) assay in minimal medium grown Legionella without, with 1 mM and with 2 mM of TPP. BlaM activity in 10μg total protein of wt and csrA- strain containing the 5'UTR of the thi-operon in a pXDC61 plasmid was measured. Each value represents the mean +/- SD of three independent experiments. BlaM activity is significantly decreased in the mutant at the different conditions indicating a positive effect of CsrA on the thi-operon expression in L. pneumophila. D) Model of the TPP riboswitch modulated by CsrA. Mfold prediction of the secondary structure of the 5'UTR thi-region: When TPP is bound, the OFF state of the riboswitch is favored in which the expression of the operon is inhibited (most likely due to premature termination at the predicted termination site). The presence of CsrA in contrast might stabilize the ON state where the structure of the thi-element is dispersed, hence, higher amounts of TPP would be necessary to shift the element back to the OFF state leading to the down-regulation of the thi-genes expression.