Role of nine repeating sequences of the mini-F genome for expression of F-specific incompatibility phenotype and copy number control

Abstract

An autonomously replicating 2,248-base-pair DNA segment of the mini-F plasmid carries nine 19-base-pair repeating sequences. Five of the repeats are arranged in one direction and form the right cluster, whereas the remaining four repeats are arranged in the opposite direction and form the left cluster (Murotsu et al., Gene 15:257-271, 1981). Each cluster, cloned separately into the multicopy plasmid vector pBR322, exhibited a strong F-specific incompatibility phenotype (FIP). These clusters were thought to be responsible for the expression of IncB and IncC phenotypes, causing a switchoff function on mini-F replication. Mini-F DNA fragments containing two, three, or more than four repeats were inserted into pBR322. Cells carrying these recombinant plasmids exhibited, respectively, no, intermediate, and strong FIP intensity. Cloning of five repeats into pSC101, whose copy number is about 6 in contrast to 20 for pBR322, resulted in an FIP of intermediate intensity. Thus, the intensity of FIP reflects the dosage of repeats in a cell. The five repeats in the right cluster were eliminated from the mini-F derivative without impairing its autonomous-replicating ability (Bergquist et al., J. Bacteriol. 147:888-889, 1981; Kline and Palchavdhuri, Plasmid 4:281-289). Such deletion, however, caused a sixfold elevation of the copy number. When the eliminated cluster of repeats was reinserted in the derivative, the copy number was lowered to the original value, viz., 1 to 2. The position and orientation of this insertion was not important in the copy number control. Thus, the repeats are also related to copy number control. A model to account for the role of the repeating sequences in the control of copy number and FIP is discussed.