Experimental evolution using Pseudomonas fluorescens SBW25 and its plasmid pQBR103 to investigate costs of plasmid carriage and compensatory mutations to ameliorate these costs

Plasmids are widespread and plasmid conjugation is the main mechanism of horizontal gene transfer driving genomic diversity in bacteria. Nevertheless, the very existence of plasmids presents a paradox. Theory predicts that irrespective of their net fitness effects on bacteria, plasmids should be lost from populations: When plasmids are parasitic (costs outweigh benefits), at realistic rates of horizontal transfer they should be lost to purifying selection, yet under mutualism (benefits outweigh costs), selection favours the capture of accessory genes by the host chromosome and loss of the plasmid backbone. Using experimental evolution of Pseudomonas fluorescens and its mercury resistance plasmid, pQBR103, we show that compensatory evolution can solve the plasmid paradox across the entire parasitism-mutualism continuum by rapidly ameliorating the cost of plasmid carriage. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Remarkably, a knockout of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Gene expression analysis revealed that mutations in gacA/gacS were likely to have relieved the elevated cellular translational demand caused by the plasmid. Rapid, parallel compensatory evolution via single regulatory mutations of large effect in bacteria can stabilise plasmids irrespective of the immediate net fitness effect that plasmid acquisition had upon the host cell. Consequently, compensatory evolution can explain the widespread occurrence of plasmids and allow horizontally acquired traits to be retained by bacteria even in environments where these genes are not immediately useful.