Figure 3.

Mutagenesis of CsrA and Hfq interacting sequences in the sepL 5′ UTR. (A) the predicted secondary structure of sepL 5′ UTR and 5′ CDS on the left-hand side of the panel shows the two AGGnA sequences predicted to interact with CsrA. The Hfq binding peaks, deletions, and ARN5m2 motif are as indicated in Figure 1. A mutation (H1) was introduced into the ARN5m2 motif to disrupt Hfq interactions at this site (peak 703, Figure 1). Four site-specific mutations (C1–4) were introduced to test their impact on CsrA activation of sepL translation while C5 was introduced to restore base pairing in SL4 that may be destabilised by the C4 mutations. (B) Measurement of the wild type and mutated SepL-GFP fusions in either the E. coli O157:H7 strain ZAP193 (high T3 secretor background) or strain Sakai (low T3 secretor). Fluorescence was measured for cultures over a range of optical densities and then adjusted for optical density as described in Materials and Methods. (C) Flow cytometry analysis of SepL-GFP and C1–4 mutations in E. coli O157:H7 ZAP193 (high T3 secretor). Expression of the wild type fusion in the ΔcsrA background indicates minimal levels of secretion in the absence of translational activation. Significance was calculated using a paired t-test for replicate data collected over multiple experiments (panel B, ZAP193 sepL-C4, -C5 and -C4&5). An unpaired t-test was used for all other samples.