Hypermutator strains of Pseudomonas aeruginosa bypass genetic constraints in the phenotypic switching triggered in biofilms

TOBARES RA; MOYANO AJ; COLQUE CA; SMANIA AM

Buenos Aires

Congreso; LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2017

Pseudomonas aeruginosa (PA) grows as biofilm communities in the airways of cystic fibrosis patients, where small colony variants (SCVs) are frequently observed. These biofilm-adapted SCVs show a great instability when grown outside biofilms, thus revealing the ability to switch between phenotypes. Hypermutator Mismatch Rrepair System (MRS)-deficient strains of PA show an increased phenotypic diversification, particularly in biofilms. Thus, we performed an evolutionary assay to explore the adaptive potential of PA and the role of hypermutability in this phenotypic switching. We founded five lines with the wt strain and four lines with its isogenic hypermutator mutS mutant, and subjected them to alternating conversion and reversion cycles, from which we further isolated SCVs and revertant clones from both strains. We next performed whole genome sequencing using an Illumina platform to carry out a comparative genomic analysis of both parental strains as well as endpoint clones of all wild type lines and intermediate clones of three mutS lines. All wt lines of PA showed the accumulation of 1 mutation/round in genes of the wsp and yfi systems, both being regulators of the second messenger c-di-GMP, which governs the transition between planktonic and biofilm mode of growth. By round 4, SCV conversion was dramatically constrained in the wt strain. On the other hand, conversion/reversion rounds continued steadily in all mutator lines, showing an average of 20 mutations/cycle. Interestingly, hypermutators also showed 1 mutation/cycle in the wsp and yfi systems, indicating that this evolutive parallelism was maintained even in the mutator lines. Apart from these, at least other 8 c-di-GMP-related genes were found to be mutated, which is still insufficient to explain the switching process carried out by mutators. Fulfilling this study will reveal new adaptive pathways explaining the ability of hypermutators to bypass the genetic constraints in phenotypic switching.