Evolution of Transcriptional Regulatory Networks in Pseudomonas aeruginosa During Long Time Growth in Human Hosts

Abstract

Bacteria are remarkable organisms with the capacity to adapt to new environments by remodelling their gene expression profiles. The specific genomic material of any bacterium determines its capacity for any gene regulatory repertoire. However, by evolutionary shaping, these regulatory networks are subjected to forces that allow the bacteria to break genomic constraints, remodel existing regulatory networks, and colonise new environments. While experimental evolution studies have documented that global regulators of gene expression are indeed targets for adaptive mutations, it is less clear to which extent these observations relate to natural microbial populations. The focus of this thesis has been to study how regulatory networks evolve in natural systems. By using a particular infectious disease scenario (human associated persistent airway infections caused by the bacterium Pseudomonas aeruginosa) as a natural model system, the work has focused on characterising a number of mutations in global regulators that are known to provide an adaptive advantage in this specific environment. The aim has been to provide a molecular explanation of the effects of the specific mutations in relation to regulatory network remodelling, and to provide insight into the extent of epistasis and evolutionary dynamics of these systems. The two studies presented in this thesis specifically deal with single amino acid substitutions or deletions in the sigma factors RpoD, AlgT, and RpoN. Through in vitro techniques, we characterised the direct molecular effects of the sigma factors’ abilities to interact with DNA and the core RNA polymerase (RNAP). By combining this approach with in vivo transcription profile data, Chromatin Immunoprecipitation-sequencing (ChIP-seq) data and artificial regulatory network modifications by in vivo sigma factor overexpression, we were able to investigate how the altered molecule-to-molecule interactions induce rewiring of transcriptional regulatory networks and create unexpected phenotypes. The results show that through remodelling of the respective regulatory networks, mutations fixed in global regulator genes facilitate the generation of novel phenotypes which again facilitate the shift in life-style of the bacterium from an environmental opportunistic pathogen to a human airway specific pathogen. These findings are not only applicable to P. aeruginosa specific studies, but suggest that, on a general level, evolutionary remodelling of regulatory network structures may be the key to ecological success in the wild.