Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli

Abstract

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did 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.

The absence of effect of gid or mioC transcription on the initiation of chromosomal replication in Escherichia coli

“…The DnaA box ⌬R4 mutant requires transcription from at least one of the promoters, possibly due to a reduced ability to melt the 13-mer region when fewer DnaA-binding sites are available. This hypothesis is consistent with findings that overexpression of DnaA protein results in significant replication in the presence of rifampicin (35,36). We propose that gidA and mioC transcription becomes essential only when oriC is under suboptimal conditions.…”

Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r

“…Marker frequency analysis of the origin to terminus ratio by hybridization is another method, which gives very precise measurements of the C period (Atlung & Hansen, 1993). However, this method is especially good for determining the C period in fast-growing bacteria where the origin to terminus ratio is high.…”

“…The C period has been determined from the residual DNA synthesis obtained after inhibition of initiation of chromosome replication (Bipatnath et al, 1998;Churchward & Bremer, 1977); from marker frequency analysis determining the origin to terminus ratio (Atlung & Hansen, 1993;Yoshikawa & Sueoka, 1963); or from determinations of the fraction of replicating cells in slowly growing bacterial cultures (Kubitschek & Newman, 1978). The D period has been reported to be constant in E. coli B/r at different generation times, estimated either from residual division in the presence of chloramphenicol (Kubitschek, 1974), or from residual division during thymine starvation (Bremer & Chuang, 1981).…”

Six GTP-binding proteins of the Era/Obg family are essential for cell growth in Bacillus subtilis b bThe primer sequences used for PCR in this study are shown as supplementary data on Microbiology Online (http://mic.sgmjournals.org).

“…Both positive and negative regulators of replication initiation have been reported previously in E. coli. An overproduction of the replication initiator protein DnaA resulted in overinitiation (Atlung & Hansen, 1993). Sequestration of newly duplicated hemimethylated origins into the cell membrane by the SeqA protein (Lu et al, 1994 ;Onogi et al, 1999) and inactivation of the DnaA protein by the DnaN and Hda proteins (Katayama et al, 1998 ;Kato & Katayama, 2001) are known negative regulatory mechanisms for suppression of initiation.…”