Flemming G. Hansen

The DnaA protein determines the initiation mass of Escherichia coli K-12

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, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

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 states could thus be followed in vitro as well as in vivo by non‐invasive fluorimetric measurements. The 1.5 Å crystal structure of the oxidized protein revealed a disulfide bond‐induced distortion of the β‐barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state‐dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox‐active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.

Autoregulation of the dnaA gene of Escherichia coli K12

SummaryRegulation 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 fusion was inhibited up to sevenfold by surplus dnaA protein and was stimulated up to fivefold upon thermoinactivation of the mutant protein in five different dnaA(Ts) strains. The autoregulation was found to be exerted at transcription from the major dnaA promoter and was eliminated by deletion of sequences around position-65 of this promoter where a 9-bp sequence, which is also found four times in the chromosomal origin, is located.

The initiator titration model: computer simulation of chromosome and minichromosome control.

The initiator titration model was formulated to explain the initiation control of the bacterial chromosome. In particular, features concerning the replication behaviour of minichromosomes, such as their high copy number and Escherichia colis ability to coinitiate chromosome and many minichromosome origins, were considered during the formulation of the model. The model is based on the initiator protein DnaA and its binding sites, DnaA boxes, in oriC, in the dnaA promoter and at other positions on the chromosome. Another important factor in the model is the eclipse period created by the hemimethylation of a new oriC which makes it refractory to initiation. The model was analysed by computer simulations using a stochastic approach varying the different input parameters, and the resulting computer cells were compared with data on living E. coli cells. Here we present the outcome of a few of these simulations concerning the eclipse period, in silico-shift experiments blocking initiation or elongation of replication, and introduction of minichromosomes into the computer cells. We also discuss the synthesis of DnaA protein in the computer cells. From our simulations, we conclude that, whether true or not, the model can mimic the in vivo initiation control of E. coli.

The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves

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 chromosome lie in the opposite half. This is achieved by reorganizing the chromosome arms of the two nucleoids in pre‐division cells relative to the cell quarters. The spatial reorganization of the chromosome arms ensures that the two replication forks remain in opposite halves of the cell during replication. The relative orientation of the two reorganized nucleoids in pre‐division cells is not random. Approximately 80% of dividing cells have their nucleoids oriented in a tandem configuration.

Progressive segregation of the Escherichia coli chromosome

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 pattern is one of continuous segregation during replication and is not consistent with recently published models invoking extensive sister chromosome cohesion followed by simultaneous segregation of the bulk of the chromosome. The terminus, and a region immediately clockwise from the origin, are exceptions to the overall pattern and are subjected to a more extensive delay prior to segregation. The origin region and nearby loci are replicated and segregated from the cell centre, later markers from the various positions where they lie in the nucleoid, and the terminus region from the cell centre. Segregation appears to leave one copy of each locus in place, and rapidly transport the other to the other side of the cell centre.

Role of SeqA and Dam in Escherichia coli gene expression: A global/microarray analysis

High-density oligonucleotide arrays were used to monitor global transcription patterns in Escherichia coli with various levels of Dam and SeqA proteins. Cells lacking Dam methyltransferase showed a modest increase in transcription of the genes belonging to the SOS regulon. Bacteria devoid of the SeqA protein, which preferentially binds hemimethylated DNA, were found to have a transcriptional profile almost identical to WT bacteria overexpressing Dam methyltransferase. The latter two strains differed from WT in two ways. First, the origin proximal genes were transcribed with increased frequency due to increased gene dosage. Second, chromosomal domains of high transcriptional activity alternate with regions of low activity, and our results indicate that the activity in each domain is modulated in the same way by SeqA deficiency or Dam overproduction. We suggest that the methylation status of the cell is an important factor in forming and/or maintaining chromosome structure.

Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in Escherichia coli

SummaryIncreased synthesis of DnaA protein, obtained with plasmids carrying the dnaA gene controlled by the heat inducible λpL promoter, stimulated initiation of replication from oriC about threefold. The overinitiation was determined both as an increase in copy number of a minichromosome and as an increase in chromosomal gene dosage of oriC proximal DNA. The additional replication forks which were initiated on the chromosome did not lead to an overall increase in DNA content. DNA/DNA hybridization showed an amplification encompassing less than a few hundred kilobases on each side of oriC. Kinetic studies showed that the overinitiation occurred very rapidly after the induction, and that the initiation frequency then decreased to a near normal frequency per oriC. The results indicate that the DnaA protein is one important factor in regulation of initiation of DNA replication from oriC.

Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli.

The organization of six open reading frames which were deduced from the nucleotide sequence of some 10 kb from the replication origin region of Bacillus subtilis resembles the organization of the genes in the rnpA‐dnaA‐gyrB region of the Escherichia coli chromosome. Based on the detection of homology with the E. coli genes the open reading frames were found to represent the Bacillus ‘rnpA’, ‘rpmH’, ‘dnaA’, ‘dnaN’, recF and gyrB genes. Only the latter two have also been defined by genetic analysis. Two regulatory regions containing nine and four copies of a repeating sequence, DnaA‐box, which is identical with the DnaA protein‐binding sequence repeated four times in the E. coli origin of replication, flank the ‘dnaA’ gene of B. subtilis. One or both of them are proposed to function as origins in the initiation of chromosomal replication. Transcription of the ‘dnaA’ gene of Bacillus starts in one of these regions and appears to be coupled to initiation of chromosomal replication. We propose that the conserved gene organization in the ‘dnaA’‐‘gyrB’ region of B. subtilis is representative of the replication origin region of a primordial replicon. The oriC sequence of E. coli has either been translocated to its present location 44 kb away from the primordial origin or has independently evolved there.

Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli Mapped by Tn 10 insertion mutations

SummaryTransposon Tn10 insertion mutations in the oriC (replication origin) — atp (operon for the subunits of the membrane-bound ATP synthase) region of the Escherichia coli K12 chromosome were used to define promoters of the atp operon. The Tn10 insertions were first isolated and physically mapped on the specialized transducing phage λasn20 with a genome size of 23.5 MD, the insertion into which of the 6 MD Tn10 did not reduce packaging or stability. After transfer by recombination into the chromosome, a class of six Tn10 insertions was found to reduce growth yield and expression of the atp operon determined by quantitizing synthesis of the c-subunit of the ATP synthase. These six Tn10 insertions mapped within a 400 basepair segment of chromosomal DNA in front of the atpB gene. This segment contains the coding sequence for a 14KD polypeptide designated atpI. Complementation tests showed that the mutations are cis dominant and revealed that the 14 KD atpI gene product is not essential for biosynthesis and activity of the membrane bound ATP synthase. The insertions appear to block transcription into the atp genes located downstream. The insertions had different effects on atp operon expression: the ones closest to the atpB gene gave a 80%–90% decrease in c-subunit synthesis while those 150–200 basepairs further upstream only gave a 60%–70% decrease. These differential effects are taken to indicate the presence of three transcription start sites for the atp operon, a major promoter, designated atpIp, in front of the atpI gene and two minor ones, atpB1p and atpB2p, within the coding sequence atpI in front of the atpB gene. The allocation of atpIp coincides with potential transcription start signals found by DNA sequence analysis.