Whole genome sequencing and transcriptomics analysis of PAO1 and strains with mutations in mexT

Pseudomonas aeruginosa, a common environmental organism, can adapt to survival in the human host as an opportunistic pathogen. Infection with P. aeruginosa (e.g. in the lung, on medical devices or in a diabetic ulcer) often leads to a clinical crisis which does not respond to management with antibacterial chemotherapy. Two well-described variants co-exist: drug susceptible (like PAO1), and drug resistant (nfxC-type mutant). The major genotypic difference between resistant and susceptible variants is a change in the copy number of an 8bp repeat within the coding sequence of the transcriptional regulator mexT, a recognised mutational hotspot. To define this adaptive mode of survival we generated, from a parent strain (PA), two mutants with one repeat deleted: an isogenic engineered mutant (PA∆), and an nfxC-type mutant selected on ciprofloxacin (PAn). The mutants were investigated using phenotypic microarrays and RNAseq. The experiments revealed that clinical adaptation is likely due to differences in virulence, chemotaxis, motility and central metabolism linked to the expression of antibiotic resistance displayed by variants with one repeat in mexT. The mechanism for this appears to be the extension of a helix-turn-helix motif, spanning the repeat region, which increases the expression of a downstream LysR region via auto-regulative mechanisms. We also identified putative compensatory mutations associated with differences in phenotype between the genetically engineered PA∆ and antibiotic selected PAn. This serves as a cautionary warning to fully understand the genetic background in which clinically important mutations are considered and investigated.