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Regulation of the dnaA gene, which codes for an essential factor for the initiation of replication from the chromosomal origin, was studied in vivo using transcriptional and translational gene fusions. We found that the dnaA gene was autoregulated over a 30-fold range by the activity of dnaA protein. Expression from the dnaA promoter region of a dnaA″lacZ(More)
To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox(More)
We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth temporal progression from origin to terminus. Thus, the overall(More)
When an E. coli mutant (CRT46, dnaA46), thermosensitive in the initiation of DNA replication, grows at intermediate temperatures its DNA/mass ratio is somewhat lower than normal, but the cells possess an excess of initiation capacity, which can be expressed in the absence of proteins synthesis and lead to the accumulation of anomalously high amounts of DNA.(More)
A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant λ and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508,(More)
DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size,(More)
We have developed a system for the simultaneous labelling of two specific chromosomal sites using two different fluorescent ParB/parS systems. Using this, we demonstrate that the two chromosome arms are spatially arranged in newborn cells such that markers on the left arm of the chromosome lie in one half of the cell and markers on the right arm of the(More)
The rnpA gene, coding for the protein component of ribonuclease P (RNase P), was allocated to the dnaA region at 83 min of the E. coli K-12 map. This was accomplished through analysis of recombinant pBR322 plasmids, some of which complemented the temperature sensitivity of a strain carrying the rnpA 49 allele and restored the RNA processing activity.(More)
The nucleotide sequence was determined of a 945-bp EcoRI fragment from the Escherichia coli K-12 chromosome at 82 min containing the promoter region of the dnaA gene. This nucleotide sequence contained a coding sequence identical to the amino acid sequence of the ribosomal protein L34 , designated rpmH . The rimA mutation, which affects the maturation of(More)
A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to(More)