Rewiring of global regulatory networks drives the adaptation of Pseudomonas aeruginosa to the airways of Cystic Fibrosis Patients

Evolutionary adaptation to novel environments often involves mutations that modify exciting regulatory networks. By whole-genome sequencing of a highly successful Pseudomonas aeruginosalineage (DK2) causing life-long infections in a group of Danish cystic fibrosis (CF) patients, we have identified mutations in the genes mucA (frame-shift), algT (base substitution), rpoN (base substitution) and lasR (deletion), all encoding important regulatory proteins. To analyze the exact phenotypic effects of these genetic changes, we moved each mutation alone and in combinations into the chromosome of the laboratory reference strain P. aeruginosa PA01 by allelic replacement. The deletion in mucA caused overproduction of alginate, but subsequent introduction of the algT allele altered the alginate regulation in a conditional manner leaving the mucA algT mutant mucoid only when cultivated under stressful conditions such as high osmolarity and anaerobic growth. Introduction of the rpoN mutation significantly reduced flagella and pili mediated motility of the PA01 derived mutant without the significant growth and metabolic defects otherwise associated with rpoN knockout mutations. To investigate epistatic interactions between the four regulatory mutations from the DK2 lineage, we constructed a quadruple mutant (Q-mutant) by introducing a lasR knockout mutation to the PA01 derived mucA algT rpoN mutant. Interestingly, the Q-mutant showed significantly increased resistance/tolerance towards beta-lactams (Ceftazidime), and aminoglycosides (Tobramycin). Transcriptomic profiling of cultured Q-mutant cells suggested multiple epistatic effects of the regulatory mutations, and we hypothesize that such interactions combined with the individual effects of the specific mutations could facilitate the adaptation ofP. aeruginosa to the CF lung environment. We further hypothesize that regulatory network rewiring may be an important adaptive mechanism behind significant life-style changes.