Mutational and in vivo methylation analysis of F-factor PifC protein binding to the pif operator and the region containing the primary origin of mini-F replication.

We have used in vivo methods to identify multiple DNA-binding sites for the negatively autoregulated mini-F replication factor PifC. Sequence analysis of pif operator constitutive mutants, isolated as insensitive to repression by PifC, establishes the structure of pifO. This site contains a 17-base-pair (bp) region of dyad symmetry with 7-bp perfect inverted repeats separated by 3 bp. In vivo DNA methylation studies with dimethyl sulfate show that the reactivity of five of six guanine residues in the pifO region is altered in the presence of PifC protein. In addition, there are several sites of PifC-dependent methylation enhancement and protection upstream of pifO within repeated sequences bearing homology to pifO. The significance of the repeated PifC binding sequences and their relationship to the primary origin of mini-F replication (oriV1) are discussed.