One of the evolutionary strategies allowing organisms to survive in fluctuating environments is the generation of phenotypic variants and subsequent selection of those adapted phenotypes. Pseudomonas aeruginosa is able to live under communities known as biofilms and it is noted that during biofilm growth it diversifies into different niche specialists. Small Colony Variants (SCV) is one of these adapted phenotypes which have been characterized as hiperadherent cells, highly biofilms producers, with diminished capability of movement. When SCV are grown on solid media, the wild type (WT) phenotype emerges from the edges of the colony indicating a switching between to phenotypic phases. However, molecular basis leading to SCV conversion and reversion remain unknown. Several reports have proposed that adaptive strategies might be accelerated in mutators, giving advantages in environments in which new phenotypes are being continually selected for, through the beneficial mutations that they may generate. In this work we analyzed the adaptive capability of P. aeruginosa when is subjected to controlled, successive and repeated rounds of SCV conversion/reversion by exposing bacteria to alternating cycles of biofilm growth (conversion) and growth in solid media (reversion). For this, five lines of mutators mismatch deficient strains (mutS mutants) and five lines of WT strains were subjected to twenty consecutive rounds of SCV conversion/ reversion. From each round, SCV or reverted WT single clones were selected and propagated in the next generation, leading to the application of bottleneck in each stage. A monitoring of SCV and reverted isolated phenotypes from initial and final rounds were further performed for biofilm production, flagella swarming mediated motility and autoaggregative behavior. Notably, all WT lines lead to a premature “evolutionary dead-end” due to the inability to SCV conversion or reversion, a phenomenon that was only observed in one of the five mutators lines. When grown in biofilms, mutator diversified into a wider variety of morphotypes whereas WT diversified almost exclusively into SCV. Furthermore, reversion process was significantly accelerated in mutators throughout the entire experiment. Our results suggest that the phenotypic switching involving SCV are due to mutational events that would be accelerated in mutator strains. Also, despite a higher mutation rate that lead to a higher accumulation of mutations, the SCV switching was reproducibly and successively produced from the P. aeruginosa mutS mutants which, in front to particular but fluctuating environmental conditions, maintained the capability to diversify along the entire evolutionary process. Whole genome sequencing of ancestral and final clones is being performed in order to evaluate those mutations which are involved in this adaptation process.
