Masamichi Kohiyama

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo.

The initiation mess

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the ‘initiation mass’. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co‐operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC ) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective pre‐initiation DnaA–oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli

A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

Co‐ordination between membrane oriC sequestration factors and a chromosome partitioning protein, TolC (MukA)

oriC DNA in the hemimethylated (but not in the fully methylated) state reacts with an Escherichia coli K‐12 outer membrane preparation. This reaction is drastically reduced when the membrane preparation of a seqA null mutant is used. An in vitro reconstitution of the activity was undertaken by adding a partially purified SeqA protein to a seqA mutant membrane without success. A possible reason for this failure might be a profound modification of the outer membrane of the seqA mutant (as revealed by the fact that membrane from the mutant sediments more slowly than that from the wild type during ultracentrifugation). There is also a reduction in the content of OmpF protein. Moreover, one of the minor outer membrane proteins involved in partitioning of newly synthesized chromosomes, the TolC (MukA) protein, was also found to be downregulated in the seqA mutant. This is also true of the hobH mutant grown in a high‐osmolarity medium. Mutants of both seqA and hobH stop dividing after hyperosmotic shock, forming filaments (as observed in dam mutants).

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K+ up to 3.6 M. This creates a very large K+ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K+ but the residual K+ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K+ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a Km of between 1 to 2 mM. The driving force for K+ transport has been explored. Internal K+ concentration is not in equilibrium with ΔΨm suggesting that K+ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N′-dicyclohexylcarbodiimide inhibits by 100% K+ transport even though membrane potential ΔΨm is maintained under these conditions. By contrast, the necessity of a ΔΨm for K+ accumulation has not yet been clearly demonstrated. K+ transport in H. volcanii can be compared with K+ transport via the Trk system in Escherichia coli.

INTERACTION OF DNAK WITH NATIVE PROTEINS AND MEMBRANE PROTEINS CORRELATES WITH THEIR ACCESSIBLE HYDROPHOBICITY

Molecular chaperones are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. They interact specifically with hydrophobic sequences that are exposed in unfolded proteins, and buried in native proteins. We have studied the interaction of DnaK with native water-soluble proteins and membrane proteins. DnaK-native protein interactions are characterized by dissociation constants between 1 and 50 microM (compared with 0.01-1 microM for unfolded proteins). This affinity is within the range of most intracellular protein concentrations, suggesting that DnaK interacts with a greater number of native proteins than previously suspected. We found a correlation between the affinity of native proteins for DnaK and their affinity for hydrophobic-interaction chromatography adsorbents, suggesting that DnaK interacts with exposed hydrophobic groups in native proteins. The interaction between DnaK and membrane proteins is characterized by DnaKs high affinity for detergent-solubilized membrane proteins, and its lower affinity for membrane proteins inserted in lipid bilayers, suggesting that the chaperone can interact with the hydrophobic sequences of the former, while it cannot penetrate the hydrophobic core of lipid bilayers. Thus, the specificity of DnaK for hydrophobic sequences is involved in its interaction with not only unfolded proteins, but also native water-soluble proteins and membrane proteins. All proteins interact with DnaK according to their exposed hydrophobicity.

Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli

To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.

Isolation of a New Hemimethylated DNA Binding Protein Which Regulates dnaA Gene Expression

In this report, we show that yccV, a gene of unknown function, encodes a protein having an affinity for a hemimethylated oriC DNA and that the protein negatively controls dnaA gene expression in vivo.

Replication cycle dependent association of SeqA to the outer membrane fraction of E. coli.

The hemimethylated oriC binding activity of the E. coli heavy density membrane fraction (outer membrane) was investigated by DNase I footprinting experiments using membranes obtained from different replication stages of PC-2 (dnaCts) cells. The maximal binding activity was found at the beginning of replication cycle and then decreased gradually. The same pattern of variation was observed with SeqA protein detected in the membranes by immunoblotting. Both binding activity and the presence of SeqA were conserved in the outer membrane even after floating centrifugation of the heavy density membrane fraction in a sucrose gradient, indicating that SeqA in fact can associate with the membrane and that this association varies according to replication cycle. Site specific binding to hemimethylated oriC, of the heavy density membrane obtained from seqA mutant, could be restored by addition of a low amount of His-tagged SeqA protein.

Hemi-methylated oriC DNA binding activity found in non-specific acid phosphatase

The lacZ–hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi‐methylated binding activity. We have purified the protein encoded by this locus to near homogeneity. The protein corresponds to the monomeric form of a non‐specific acid phosphatase (NAP) whose gene has been designated aphA. oriC DNA footprinting experiments showed protection of hemi‐methylated probe by partially purified NAP, but not by purified preparations. Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg2+. This experiment also showed an increased affinity of the protein for the hemi‐methylated probe compared with the fully or unmethylated form. Indirect immunofluorescence microscopy revealed the existence of discrete NAP foci at mid‐cell in cells with two nucleoids, but at cell poles in those with one nucleoid.