Fig. 4

A Intracellular survival of the wild type and ?flrA in A. castellanii calculated by dividing the number of bacteria that were detected at 4 h by the 2 h samples. B CI of the wild type and ?flrA in A. castellanii and LB (C) calculated by dividing the output ratio (WT/?lacZ) after incubation corrected by the input ratio. D Swimming motility of the wild type and ?flrA expressed as diameter (mm) of the zone travelled from point of inoculation by bacteria grown overnight on semi-solid LB agar plates. E Biofilm biomass of the wild type and ?flrA quantified by CV staining. F Haemolytic activity of the wild type and ?flrA expressed as percent haemolysis of 1% sheep erythrocyte suspension. G Protease activity in culture supernatants of the wild type and ?flrA measured by azocasein hydrolysis assay. H Swimming motility of the wild type and ?flrA complemented with either pBAD24 or pBAD24 containing wild type flrA and/or mutated copies of the flrA (A213V and V261G). Values are expressed as diameter (mm) of the zone travelled from point of inoculation by bacteria grown overnight on semi-solid LB agar plates containing ampicillin and with or without arabinose. All of the data are from nine independent biological replicates and are shown as the mean ± standard deviation. Statistical significance was determined by two-tailed, non-parametric Mann?Whitney test (A?G), one-way ANOVA and Kruskal?Wallis test (H) and is indicated by ^???p < 0.001. I Scanning electron micrograph showing presence or absence of flagellum on V. cholerae. Wild type A1552 (top left), A1552 ?flrA (top right), A1552 flrA A213V (Bottom left), A1552 flrA V261G (Bottom right). Presence of flagella in wild type image indicated with black arrow. Scale bars: 1 µm. J Principal component analysis (PCA) on the changes in the four virulence phenotypes (motility, biofilm, hemolysin and protease) across adapted isolates, non-adapted isolates, wild type and ?flrA mutant.