Copy Number of ColE1-type Plasmids Decreases When rop is Supplied in trans on a Separate Plasmid

Previous studies have shown that in Escherichia coli co-transformed with two different plasmids, the expression of the Rop protein from one plasmid can act in trans to decrease the copy number of the other plasmid, an effect referred to as plasmid exclusion. These studies relied on previous findings on ColE1-type plasmids showing that copy number is primarily regulated by the RNA II transcript, which serves as a primer for plasmid replication, the RNA I transcript, which binds and sequesters RNA II, and the Rop protein, which stabilizes the RNA I : RNA II hybrid. However, these studies, which used the pUC19, pBR322, and pCAWK vectors, did not account for the influence of an RNA II point mutation that was present in pUC19, but absent in both pBR322 and pCAWK. Hence, we decided to use the pAPA3 plasmid in lieu of pUC19, to control for the effect of the RNA II point mutation. We hypothesized that by controlling for the RNA II point mutation with the use of pAPA3 plasmid, the presence of Rop would result in greater levels of exclusion of the lower copy number plasmids in co-transformed cells. To test this hypothesis we generated co-transformants in Escherichia coli DH5α containing either pBR322 or pCAWK and pAPA3 plasmids. Plasmids from co-transformants were isolated and copy numbers were estimated by transforming E. coli DH5α and selecting for growth using antibiotic selection. The ratio of the number of transformants on the selective media was used as a proxy for the ratio of pAPA3 to pBR322 or pCAWK in the co-transformed cells. We observed that the pAPA3:pCAWK ratio was 6 times greater than the pAPA3:pBR322 ratio, suggesting that the expression of Rop reduces plasmid exclusion. Based on this observation, we propose that the Rop expressed by pBR322 acted in trans to reduce pAPA3 copy numbers, effectively increasing the ratio of pBR322 relative to pAPA3 in the cell. Conversely, the absence of the rop gene on both pAPA3 and pCAWK allowed pAPA3 to replicate to a higher copy number, thereby reducing the ratio of pCAWK relative to pAPA3.