P1 plasmid replication. Role of initiator titration in copy number control

The copy number control locus incA of unit copy plasmid P1 maps in a region containing nine 19 base-pair repeats. Previous results from studies in vivo and in vitro indicated that incA interacts with the plasmid-encoded RepA protein, which is essential for replication. It has been proposed that the repeat sequences negatively control copy number by sequestering the RepA protein, which is rate-limiting for replication. Our results lend further support to this hypothesis. Here we show that the repeats can be deleted completely from P1 miniplasmids and the deletion results in an approximately eightfold increase in plasmid copy number. So, incA sequences are totally dispensable for replication and have only a regulatory role. The copy number of incA-deleted plasmids can be reduced if incA sequences are present in trans or are reincorporated at two different positions in the plasmid. This reduction in copy number is not due to lowered expression of the repA gene in the presence of incA. We show that one repeat sequence is sufficient to bind RepA and can reduce the copy number of incA-deleted plasmids. When part of the repeat was deleted, it lost its ability to bind as well as influence copy number. These results show a strong correlation between the capacity of incA repeats to bind RepA protein both in vivo and in vitro, and the function of incA in the control of copy number.