Cyclic-di-GMP signalling mutants drive ecological succession and self-generated diversity in experimentally evolved biofilms of Pseudomonas aeruginosa

Biofilms represent a discrete form of microbial life which are physiologically distinct from free-living planktonic cells. The altered phenotypic manifestations of the biofilm may also elicit lifestyle-dependent adaptive responses to selective pressures. In this work, an experimental evolution model was used to study the adaptation to a biofilm lifestyle in <i>Pseudomonas aeruginosa</i> PA14. The serial passage of biofilms selected for biofilm hyperproduction in a stepwise fashion characterized by increased biomass production and phenotypic diversification was not associated with reduced susceptibility to antibiotics. Adaptation to a biofilm lifestyle selected for mutations causes constitutive increases of intracellular c-di-GMP concentrations via mutations in the phosphodiesterase <i>dipA</i>, the <i>yfiBNR</i> signalling complex and the bifunctional diguanylate cyclase/phosphodiesterase <i>morA</i>. Furthermore, selection for biofilm hyperproduction also gave rise to self-generated diversity by eliciting morphotypic diversification into complex community structures. Individual morphotypes were not associated with specific mutations and lineages dynamically switched between morphotypes despite possessing conserved mechanisms of biofilm hyperproduction. This work provides insights into the evolutionary importance of self-generated diversity to the biofilm and reveals the genetic control and phenotypic dynamics which contribute to the characteristically rugged fitness landscape associated with a sessile lifestyle.