V. cholerae adopts several survival strategies in aquatic environments. The bacterium can survive as free-living or in association with zooplankton and can build biofilm and rugose colonies [2, 3]. Recent studies have shown that V. cholerae has an enhanced growth in association with the free-living amoeba A. castellanii at 30°C [6–10] and both these microorganisms are detected in same water samples from cholera endemic area .
In this study, the internal in-frame toxR deletion mutant V. cholerae strain succeeds to express the ToxR-repressed OmpT instead of the ToxR-activated OmpU. This altered expression of the ToxR-regulated OmpU and OmpT was confirmed by previous study and might affect the viruelence of V. cholerae.
Our paper has disclosed the role of ToxR regulatory protein in the environmental survival strategies of V. cholerae such as biofilm formation, switching from smooth to rugose colony morphotypes and association with the free-living amoeba namely A. castellanii.
Our results demonstrate that ToxR clearly affects biofilm and rugose formation since the differences in biofilm and rugose colony formation between V. cholerae wild type and toxR mutant were significantly high by t-test and χ2 test, respectively.
Effect of ToxR on growth and survival of V. cholerae associated with A. castellanii was studied by viable count performed on blood agar. V. cholerae wild type strain survived longer than the toxR mutant strain at 37°C, in the presence or absence of the amoebae. Interestingly, the association with A. castellanii enhanced survival of both bacterial strain but the wild type strain survived longer uncovering a role of ToxR in the survival of V. cholerae associated with protozoa in aquatic environment.
In cultivation with the amoebae at 25°C, it was observed that both V. cholerae wild type and toxR mutant strains survived more than 10 days.
Despite V. cholerae wild type and toxR mutant strains survived more than 10 days in cultivation with the amoebae at 25°C, these strains did not form any rugose colony indicating that the bacteria avoided starvation. However, presence of the amoebae might enrich the cultivation medium and viable count of the bacteria was performed on enriched plates (blood agar plates).
Analogies to rugosity can be found in a number of other bacterial species, including the expression of alginate by mucoid strains of Pseudomonas aeruginosa and the expression of an adhesive EPS by members of the marine genus .
Spontaneous and reversible variation in cell-associated and cell-free EPS production represents an optimal adaptive mechanism that facilitates survival in stressful environments .
Although, over 20 genes are co-ordinately controlled by the ToxR regulon , the mechanism of switching is currently under study and it seemed to be regulated by exopolysaccharide related phase variation. Phase variation can occur via DNA inversion, DNA recombination, and slipped strand mispairing and is known to be involved in controlling the expression of several surface structures of gram-negative bacteria, including fimbriae, flagella, outer membrane proteins, lipopolysaccharide, and capsular polysaccharide . However, role of ToxR was found to be critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization [11, 12]. Surprisingly, ToxR homolog from V. anguillarum was found to regulate its own production, bile resistance, and biofilm formation  which might emphasis the regulatory role of ToxR in the expression of virulence factors for Vibrio species.