Experimental Evolution of the Megaplasmid pMPPla107 in Pseudomonas stutzeri Enables Identification of Genes Contributing to Sensitivity to an Inhibitory Agent

Horizontally transferred elements such as plasmids can, at times, burden host cells with various metabolic and fitness costs. Our previous work demonstrated that acquisition of the Pseudomonas syringae megaplasmid pMPPla107 causes sensitivity to a growth inhibiting substance that is produced in cultures during growth under standard laboratory conditions. After 500 generations of laboratory passage of P. stutzeri lines containing pMPPla107, two out of six independent lines displayed resistance to this inhibitory agent. We therefore sequenced the genomes of isolates from each independent evolutionary line to identify the genetic basis of this resistance phenotype through comparative genomics. Our analysis demonstrates that two different compensatory mutations on the megaplasmid ameliorate the sensitivity phenotype: 1) a large deletion of approximately 368kb in pMPPla107 and 2) a SNP in the gene we name skaA for Supernatant Killing Activity. These results provide further evidence that costs associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes.