3 DISTINCT CHROMOSOME-REPLICATION STATES ARE INDUCED BY INCREASING CONCENTRATIONS OF DNAA PROTEIN IN ESCHERICHIA-COLI

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located o n a plasmid. In these cells with different DnaA protein levels, we mea sured several parameters: dnaA gene expression; cell size, amount of D NA per cell, and number of origins per cell by flow cytometry; and ori gin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to hi gher levels of DnaA protein could be divided into three states. From t he normal level to a level 1.5-fold higher, DnaA protein had little ef fect on dnaA gene expression and the rate of DNA replication but led t o nearly proportional increases in DNA and origin concentrations. Betw een 1.5- and 3-fold, the normal DnaA protein concentration. dnaA gene expression was gradually decreased. In this interval, the origin conce ntration increased significantly; however, the replication rate was se verely affected, becoming slower-especially near the origin-the higher the DnaA protein concentration, and as a result, the DNA concentratio n was constant. Further increases in the DnaA protein concentration di d not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.