ANALYSIS OF ADAPTABILITY TO BIMODAL SWITCHING BETWEEN BIOFILM AND PLANKTONIC STATES IN MUTATOR STRAINS OF Pseudomonas aeruginosa

COLQUE CA; TOBARES RA; SMANIA AM

Córdoba

Congreso; XI Congreso de la Sociedad de Microbiología General (SAMIGE); 2015

SAMIGE

Pseudomonas aeruginosa is an opportunistic pathogen, which cause chronic biofilm infections of humans with underlying predispositions. During biofilm growth, P. aeruginosa rapidly diversifies into different niche specialists by a process known as adaptive radiation. Adaptive radiation can be easily visualized by the emergence of variants with distinct colony morphologies. Importantly, we have previously shown that high mutation rates in P. aeruginosa MRS deficient strains (mutS-) is related to an increase in phenotypic diversification, particularly during the biofilm mode of growth. Small Colony Variant (SCV) is one of these biofilm adapted phenotypes, which are characterized by a diminished capability of movement, and a hyperadherent and autoaggregative behavior. Together, these phenotypes result into high biofilm producers and very small size colonies relative to the wild-type (WT) phenotype. When SCV is grown on solid media, the WT phenotype emerges from the edges of the colony indicating a bimodal switching between two phenotypic phases. To evaluate the adaptability of P. aeruginosa, an evolutionary assay was performed by exposing the bacteria to alternating and successive rounds of SCV conversion in biofilms and WT reversion in solid media. Previous results in WT strains showed that SCV conversion and reversion involved the acquisition of mutations, mainly in wsp and yfiBNR operons, that were accumulated one per round as compensatory mutations. These genes have been recently associated to the modulation of the levels of the second messenger c-diGMP and biofilm formation. If adaptability to bimodal switching between SCV conversion in biofilms and reversion on solid media is based on the acquisition of mutations, how is this process affected by an increase in mutation rate? In this work we analyzed the genetic basis of SCV conversion and reversion in mutS- strains. First results indicate that bimodal switching between this two phases in mutS- strains was able to go on for over 16 rounds, while WT strains showed much less capacity. Then, whole genome sequencing of ancestral and final clones was carried out. Our results showed that mutS- strains have acquired a significantly higher number of mutations per round (≅19/round) over mutations seen in WT strains (1/round). The analysis of mutational spectra in mutS- strains, was strongly skewed towards the typical spectrum of MRS deficient strains, dominated by transitions and small indels. Most mutations affected genes involved in motility and attachment (pelB, wsp system, flgL, among others). However, in the first three cycles of SCV conversion/reversion, wsp and yfiBNR genes alternatively accumulated 1 mutation per round. Then, the modulation of c-diGMP levels may play a central role in the adaptation of P. aeruginosa mutS- strains-, while the rest of observed mutations could have a fine tuning or neutral role in the adaptive process as a consequence of a higher mutation rate.