A long-term experimental evolution study by comparative genomics: Modulation of c-diGMP has a key role in Pseudomonas aeruginosa adaptation to bimodal switching between biofilm and planktonic states.

Córdoba

Congreso; XI CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE; 2015

One of the most fascinating bacterial adaptive strategies during growth in fluctuating environments is phenotypic diversification into different niche specialists and subsequent selection of those adapted phenotypes by natural selection. We have been studying the opportunistic pathogen Pseudomonas aeruginosa, which is able to grow in communities known as biofilms. Biofilms can be considered as heterogeneous and stratified environments so that biofilm growth intrinsically implies phenotypic diversification. Small Colony Variants (SCV), one of the adapted morphotypes that arise from biofilm cultures, have been characterized as small size colonies formed by hyperadherent and highly biofilm producer cells with a diminished capability of movement. When SCV are grown in solid media, wild type-like (WT) morphotypes emerge from the colony edges, which show reversion of the SCV phenotypic traits. In this work, we analyzed the adaptability of P. aeruginosa using a combination of long-term experimental evolution assays and whole-genome sequencing. Three lines of P. aeruginosa UCBPP-PA14 were exposed to alternating and successive cycles of biofilm growth (conversion) and growth in solid media (reversion), and subjected to rounds of evolution in which clones with SCV or WT morphology were isolated from conversion or reversion rounds, respectively. Single SCV clones were propagated as founders in the next reversion round, as well as single WT clones were used to found the next round of conversion, leading to succeeding bottleneck events. Evolutionary histories were stopped when the frequency of emerged SCV or WT morphotypes in the respective rounds was less than 0.05%. Genomes from the ancestral and final clones obtained from each three lines were sequenced on an Illumina Hiseq1500 platform. Comparative genomic analysis demonstrated the acquisition of one non-synonymous mutation per round of evolution. Strinkingly, mutations in the three parallel evolving lines were found mostly in wsp and yfiBNR operon genes, codifying for chemosensory systems involved in regulating biofilm formation through modulation of c-diGMP levels. We hypothesize that, in our experimental evolution assay, SCV conversion and reversion switching is based on mutations that produce stimulation or inhibition of diguanilate cyclase catalyzed c-diGMP synthesis, respectively. Complementation experiments in the SCV clones obtained in the different evolving conversion rounds using the P. aeruginosa PA2133 phosphodiesterase produced a clear reversion of SCV traits, indicating the involvement of the underlying mutations in increasing c-diGMP. So far, our results indicate that, in our experimental evolutionary model, adaptability of P. aeruginosa to bimodal switching between biofilm and planktonic states is based on compensatory spontaneous mutations in genes involved in the regulation of the intracellular levels of the second messenger c-diGMP.