DnaA protein dependent denaturation of negative supercoiled oriC DNA minicircles.

The DnaA protein binds specifically and tightly to oriC supercoiled 641 bp minicircle DNA. The binding of the initiator promoted a partial unwinding of the superhelical oriC minicircle (Mc-oriC). Open complexes are detected either by a change in the linking number or by the sensitivity to the attack of a single strand specific Bal 31 nuclease. The open complex is found only in the presence of the DnaA protein.

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

BACKGROUND: 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. RESULTS: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. CONCLUSIONS: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Initiation of DNA replication in delta cya mutants of Escherichia coli K12.

The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K(A) value for cAMP as 0.819 muM(-1). The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competitive in nature with an inhibition constant (K(i)) of about 8.25 muM. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta++ cya, but also that DnaA protein is not degraded. The Delta cya mutants of E. coli are unable to continue DNA synthesis in the absence of de novo protein synthesis and the initiation of DNA replication in these mutants takes place from oriC.

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.

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 DnaK's 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.

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 immunofluorescene 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.

Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose.

The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose. Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.

High-affinity binding of hemimethylated oriC by Escherichia coli membranes is mediated by a multiprotein system that includes SeqA and a newly identified factor, SeqB.

The binding of hemimethylated oriC to Escherichia coli membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein. We describe the resolution of the membrane-associated oriC-binding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated oriC. The active component in one fraction is identified as SeqA. The active component of the second fraction is a previously undescribed protein factor, SeqB. The reconstituted system reproduced the salient characteristics of the membrane-associated binding activity, suggesting that the membrane-associated oriC-binding machinery of E. coli is likely to be a multiprotein system that includes the SeqA and SeqB proteins.

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant.

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage lambda gt11 called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997) 191-198) found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.

DnaJ potentiates the interaction between DnaK and alpha-helical peptides.

Molecular chaperones bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides, and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. The DnaK chaperone of Escherichia coli is known to interact preferentially with positively charged hydrophobic peptides in an extended conformation. Accordingly, we show in the present study that DnaK has a low affinity for alpha-helical peptides. In the presence of its co-chaperone DnaJ and ATP, however, DnaK interacts more efficiently with alpha-helical peptides. This suggests that DnaJ triggers a conformational change in DnaK which improves its interaction with these peptides. The ability of the DnaK/DnaJ/GrpE chaperone machine to interact with alpha-helical peptides (which represent the most frequent secondary structure in proteins) should be an important part of its role in protein folding and renaturation.

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 ToiC (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).

Defect in export and synthesis of the periplasmic galactose receptor MglB in dnaK mutants of Escherichia coli, and decreased stability of the mglB mRNA.

The high-affinity galactose permease, which comprises the periplasmic galactose receptor MglB, the membrane translocator MglC and the membrane-associated ATPase MglA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli. This reduced transport activity correlated with a reduction in the quantity of MglB. At 42 degrees C, an accumulation of pre-MglB in the dnaK temperature-sensitive mutant reflected a defect in MglB export. In addition, an accumulation of pre-MglB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor. At 30 degrees C, there was no accumulation of pre-MglB in the dnaK mutant, but there was still a decreased amount of MglB in the periplasm. The reduction in MglB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MglA protein (which is expressed from the same operon as MglB) was synthesized in normal amounts. Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mglBAC mRNA, and a more stable and more abundant mglB mRNA, produced by 3'-5' degradation of the mglBAC mRNA (R. W. Hogg, C. Voelker & I. von Carlowitz, 1991, Mol Gen Genet 229, 453-459). The mglB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3' extremity, which is responsible for the higher expression of MglB compared to MglA and MglC. The decreased MglB expression in the dnaK mutant at 30 degrees C in the present work correlated with a reduced stability of the mglB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mglB gene.

Specificity of DnaK for arginine/lysine and effect of DnaJ on the amino acid specificity of DnaK.

Molecular chaperones form a class of proteins that bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress. Chaperones70, including the DnaK chaperone of Escherichia coli, interact specifically with peptides enriched in internal hydrophobic residues, with a preference for positively charged peptides. We previously reported that DnaK interacts with the hydrophobic amino acids Ile, Leu, Val, Ala, Phe, Trp, and Tyr. In the present study, we show that DnaK also possesses a specific binding site for the positively charged amino acids arginine and lysine. Furthermore, the binding of arginine and lysine to DnaK is strengthened when its hydrophobic binding sites are occupied. The specificity of DnaK for Arg/Lys is supported by DnaK-peptide binding studies; the homopolypeptides poly-Arg and poly-Lys interact with DnaK, contrasting with other hydrophilic homopolypeptides, and hydrophobic peptides interact more strongly with DnaK if they contain Arg/Lys at their N terminus. Interestingly, the cochaperone DnaJ attenuates the interaction of DnaK with hydrophobic amino acids while strengthening its interaction with arginine or lysine. The interaction of DnaK with both hydrophobic sequences and with arginine and lysine, and its modulation by DnaJ, may have important implications in both protein folding and protein insertion into membranes.

A novel cytoplasmic hemimethylated oriC binding activity.

Using hemimethylated, fully methylated, and unmethylated oligonucleotide probes corresponding to part of the origin of Escherichia coli DNA replication, oriC (+81-136), we have characterized a novel hemimethylated DNA-specific protein binding activity. This activity appears to be located in the cytoplasm rather than in membrane fractions. It has been partially purified and, in DNase footprinting analysis, found to preferentially protect only a subset of the hemimethylated GATC sites present in the minimal oriC. These sites are found adjacent to the DnaA binding box, R1, and overlap the integration host factor binding site. The activity does not correspond to known hemimethylated binding proteins, although in the seqA deletion mutant, there is a 3-fold reduction of the activity. The stage of the cell cycle in synchronized PC2 cultures does not seem to significantly affect thte relative levels of this binding activity. A possible role in sequestration of the newly replicated hemimethylated origin is discussed.

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 preinitiation 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 function of Escherichia coli chaperone DnaJ. Protein-disulfide isomerase.

Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes. The DnaJ protein of Escherichia coli and the DnaJ-like proteins of eukaryotes are known as molecular chaperones and specific regulators of DnaK-like proteins and are involved in protein folding and renaturation after stress. In this study we show that DnaJ, like thioredoxin, protein-disulfide isomerase, and DsbA, possesses an active dithiol/disulfide group and catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). These results suggest that, in addition to its known function as a chaperone, DnaJ might be involved in controlling the redox state of cytoplasmic, membrane, or exported proteins.

Reversal by GroES of the GroEL preference from hydrophobic amino acids toward hydrophilic amino acids.

The chaperones GroEL/hsp60 are present in all prokaryotes and in mitochondria and chloroplasts of eukaryotic cells. They are involved in protein folding, protein targeting to membranes, protein renaturation, and control of protein-protein interactions. They interact with many polypeptides in an ATP-dependent manner and possess a peptide-dependent ATPase activity. GroEL/hsp60 cooperates with GroES/hsp10, and the productive folding of proteins by GroEL generally requires GroES, which appears to regulate the binding and release of substrate proteins by GroEL. In a recent study, we have shown that GroEL interacts preferentially with the side chains of hydrophobic amino acids (Ile, Phe, Val, Leu, and Trp) and more weakly with several polar or charged amino acids, including the strongest alpha-helix and beta-sheet formers (Glu, Gln, His, Thr, and Tyr). In this study, we show that GroES reduces the specificity of GroEL for hydrophobic amino acids and increases its specificity for hydrophilic ones. This shift by GroES of the GroEL specificity from hydrophobic amino acids toward hydrophilic ones might be of importance for its function in protein folding.

Importance of the replication origin sequestration in cell division of Escherichia coli.

The DNA adenine methyltransferase of Escherichia coli methylates adenines at GATC sequences. The mutant deficient in this methylase has no apparent deficiency in the cell division process in spite of the absence of both synchrony in initiations of chromosomal DNA replication and sequestration of replication origin (oriC) at hemimethylated state. However, the dam mutant cannot resume cell division after hyperosmotic shock differing from the wild-type strain. This inhibition is not provoked by induction of the cell division inhibitor, SfiA protein. Although the FtsZ protein is present in the dam mutant in a reduced amount compared to wild-type, the quantitative difference of this protein is not the main reason of division arrest provoked by hyperosmotic shock. This observation supports the idea of oriC-membrane interaction playing a role both in chromosome partitioning and cell division as predicted by replicon theory.

Localization of DnaK (chaperone 70) from Escherichia coli in an osmotic-shock-sensitive compartment of the cytoplasm.

The chaperone DnaK can be released (up to 40%) by osmotic shock, a procedure which is known to release the periplasmic proteins and a select group of cytoplasmic proteins (including thioredoxin and elongation factor Tu) possibly associated with the inner face of the inner membrane. As distinct from periplasmic proteins, DnaK is retained within spheroplasts prepared with lysozyme and EDTA. The ability to isolate DnaK with a membrane fraction prepared under gentle lysis conditions supports a peripheral association between DnaK and the cytoplasmic membrane. Furthermore, heat shock transiently increases the localization of DnaK in the osmotic-shock-sensitive compartment of the cytoplasm. We conclude that DnaK belongs to the select group of cytoplasmic proteins released by osmotic shock, which are possibly located at Bayer adhesion sites, where the inner and outer membranes are contiguous.

Specific interaction of the Escherichia coli chaperone GroEL (60-KDA heat shock protein) with the liganded form of the galactose binding protein.

The Escherichia coli chaperone GroEL interacts more strongly with the liganded form of the galactose binding protein (the galactose binding protein-galactose complex), than with its unliganded form. This specific interaction is reflected by the stimulation of the ATPase activity of GroEL by the liganded galactose binding protein. Interactions between native proteins and chaperones could be more frequent than generally suspected, and may help to detect protein conformational changes.