Competition between Parental and Recombinant Plasmids Affects the Measure of Recombination Frequencies

Abstract

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB ; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.