Fig. 1: CsrA forms distinct foci structures.

A Schematics of the full-length csrA gene, translationally (a) or transcriptionally (b) fused to gfp. Boxes represent translated genes, and the solid green line represents an untranslated region containing a synthetic ribosomal binding site (RBS). Both fusions are located at the native chromosomal locus of the csrA gene. Black arrows and lines represent the native regulatory region (PcsrA) and 5’UTR of csrA, respectively. B Formation of CsrA-GFP foci in EPEC. EPEC strains containing csrA-gfp and csrA-rbs-gfp (strains NE9077 and NE9121 shown in A) were grown in DMEM to OD₆₀₀ 0.5. Next, bacterial cells were fixed, washed, and visualized by fluorescence microscopy. Scale bar 2 µm. The two right panels show enlarged sections marked in the merge panels. C Quantification of CsrA foci. Bacteria were grown and fixed as in (B). The percentage of foci containing bacteria was determined by microscopy. For each experiment, n ≥ 200 bacteria were analyzed in each sample. Data presented as mean values and standard deviation of three independent experiments, with an unpaired two-tailed t-test p-value = 0.0023 (**) between the compared samples. ND not-detectable. D Distribution of focus sizes. Bacteria were grown and processed as described in (B). Analysis of CsrA-GFP foci area size was done using the NIS Elements software. In each biological repeat, n > 400 bacterial focus areas were measured. The largest focus area detected in the same biological repeat was set as 100%. The relative percentage range of the focus size is indicated in the X-axis. Data presented as mean values and standard deviation of three biological repeats. E Formation of CsrA foci by enteropathogens. Cultures of EPEC, C. rodentium (C.R.), Y. pseudotuberculosis (Y.P.), and V. cholerae (V.C.), expressing CsrA-GFP translational fusion from an inducible plasmid were grown to OD 0.45–0.6. Induction was done in the final hour of growth. In the case of S. typhimurium (S.T), csrA-gfp is translationally fused on the chromosome (strain LA11509). All samples were processed and visualized as in (B) and quantified as in (C). The Y axis represents the average percentage of focus containing bacteria and the error bars represent the standard deviation of 3 different images taken from each sample. n ≥ 200 bacteria. ND non-detectible. F Representative images of the experiment described in (E). Scale bar 2 µm.