Fig. 4.

RsmA/E binding sites in pflu3655 control capsulation. (A) Schematic diagram of pflu3655 5′-region. Two putative RsmA/E binding sites (orange squares) are located in the promoter and 5′-region of the gene. Numbers indicate nucleotide positions relative to start codon (not to scale). Sequences of putative RsmA/E binding sites are shown, the putative RBS is underlined. Gray box: Point mutations introduced in the different sequences by site-directed mutagenesis. (B) Expression of the P_pflu3655-GFP reporter carrying the different point mutations in the 1B⁴ background. GFP fluorescence was measured by flow cytometry. Data are representative of three independent experiments. (C, D) Mutations in putative RsmA/E binding sites affect capsulation in 1B⁴ (C) and SBW25 (D). Individual point mutations were reintroduced into 1B⁴ and SBW25 carrying the wild-type P_pflu3655-GFP reporter and the proportion of GFP positive cells in late exponential phase (OD_600nm = 1–2) was measured by flow cytometry. Means ± SEM are shown, n = 9 (1B⁴) or n = 7 (SBW25). Data are pooled from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskall–Wallis test with Dunn’s post hoc correction, comparison to 1B⁴. (E) Specific interaction between RsmA1 and pflu3655 mRNA. EMSA experiments were performed with 6.25 nM biotin-labelled oligonucleotides, either using a wild-type pflu3655 sequence (WT RNA; left) or a version carrying both G-8A and A33T point mutations (mut RNA; right). An increasing concentration of purified His₆-RsmA1 (RsmA1) or a high concentration of proteins purified from SBW25 expressing a non-His₆-tagged version of RsmA1 (Control protein) was added to the reactions.