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Plasmids pRAS3.1 and pRAS3.2 are natural variants of the IncQ-2 plasmid family, that except for two differences, have identical plasmid backbones. Plasmid pRAS3.1 has four 22-bp iterons in its oriV region, while pRAS3.2 has only three 6-bp repeats and pRAS3.1 has five 6-bp repeats in the promoter region of the mobB-mobA/repB genes and pRAS3.2 has only four. In previous work, we showed that the overall effect of these differences was that when the plasmid was in an Escherichia coli host, the copy numbers of pRAS3.1 and pRAS3.2 were approximately 41 and 30, respectively. As pRAS3.1 and pRAS3.2 are likely to have arisen from the same ancestor, we addressed the question of whether one of the variants had an evolutionary advantage over the other. By constructing a set of identical plasmids with the number of 22-bp iterons varying from three to seven, it was found that plasmids with four or five iterons displaced plasmids with three iterons even though they had lower copy numbers. Furthermore, the metabolic load that the plasmids placed on E. coli host cells compared with plasmid-free cells increased with copy number from 10.9% at a copy number of 59 to 2.6% at a copy number of 15. Plasmid pRAS3.1 with four 22-bp iterons was able to displace pRAS3.2 with three iterons when both were coresident in the same host. However, the lower-copy-number pRAS3.2 placed 2.8% less of a metabolic burden on an E. coli host population, and therefore, pRAS3.2 has a competitive advantage over pRAS3.1 at the population level, as pRAS3.2-containing cells would be expected to outgrow pRAS3.1-containing cells.

Evolutionary Competitiveness of Two Natural Variants of the IncQ-Like Plasmids, pRAS3.1 and pRAS3.2