Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells.

Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA-dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA-acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild-type DnaA did not. The mutations include substitutions and deletions in the phospholipid-interacting domain as well as some small deletions in the DNA-binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid- deficient cells rescued by the expression of DnaA having a point mutation in the membrane-binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild-type cells of plasmid-borne DnaA(L366K) and DnaA(Delta363-367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli.

CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.

Escherichia coli DnaA protein--phospholipid interactions: in vitro and in vivo.

DNA replication in Escherichia coli is controlled at the initiation stage, possibly by regulation of the essential activity of DnaA protein. The cellular membrane has long been hypothesized to be involved in chromosomal replication. Accumulating evidence, both in vitro and in vivo, that supports the importance of membrane phospholipids influencing the initiation activity of DnaA is reviewed.

DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane.

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.

Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.

DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replication, binding to oriC DNA, binding of allosteric effector adenine nucleotides, and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facilitate structure-function studies and mutagenic analyses of this initiator protein.

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.

The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase.

The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Membrane-mediated release of nucleotide from an initiator of chromosomal replication, Escherichia coli DnaA, occurs with insertion of a distinct region of the protein into the lipid bilayer.

DnaA protein, the initiator protein of E. coli chromosomal replication, can be rejuvenated from an inactive ADP form to active ATP-DnaA protein by acidic phospholipids in a fluid bilayer. Cross-linking studies with the photoactivable phospholipid analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H- diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine reveal insertion of DnaA protein into the hydrophobic region of the bilayer; this insertion is accompanied by membrane-mediated dissociation of the tightly bound allosteric nucleotides ADP and ATP. Photolabeling of DnaA protein occurred with membrane properties that resembled those needed for reactivation of ADP-DnaA protein; efficient labeling of DnaA protein was observed only when the lipid analog was incorporated into anionic vesicles and the temperature during treatment was above the gel to liquid crystalline phase transition. Predominant hydrophobic photolabeling was localized within a single region of DnaA protein, a region that contains putative amphipathic helices and has been shown to contain information essential for functional interaction with membranes.

The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro.

Fis protein participates in the normal control of chromosomal replication in Escherichia coli. However, the mechanism by which it executes its effect is largely unknown. We demonstrate an inhibitory influence of purified Fis protein on replication from oriC in vitro. Fis inhibits DNA synthesis equally well in replication systems either dependent upon or independent of RNA polymerase, even when the latter is stimulated by the presence of HU or IHF. The extent of inhibition by Fis is modulated by the concentrations of DnaA protein and RNA polymerase; the more limiting the amounts of these, the more severe the inhibition by Fis. Thus, the level of inhibition seems to depend on the ease with which the open complex can be formed. Fis-mediated inhibition of DNA replication does not depend on a functional primary Fis binding site between DnaA boxes R2 and R3 in oriC, as mutations that cause reduced binding of Fis to this site do not affect the degree of inhibition. The data presented suggest that Fis prevents formation of an initiation-proficient structure at oriC by forming an alternative, initiation-preventive complex. This indicates a negative role for Fis in the regulation of replication initiation.

Membrane regulation of the chromosomal replication activity of E. coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins.

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C. Whereas purified wild-type DnA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild-type protein. In a system reconstituted with purified proteins at 30 degrees C, the mutant protein initiated replication of single-stranded DNA that contains a DnA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.

DnaA protein is sensitive to a soluble factor and is specifically inactivated for initiation of in vitro replication of the Escherichia coli minichromosome.

DnaA protein loses the capacity to initiate chromosomal replication when treated with a soluble cell extract. This inactivation depends upon DNA and hydrolyzable ribonucleoside triphosphate. The extract does not affect the activities of other replicative proteins or the ability of DnaA to initiate replication of single-stranded DNA that contains a DnaA-binding hairpin, indicating that the inhibitory effect is specific for the action of DnaA at oriC. Gel filtration experiments implicate a 150-kDa factor as being responsible. Mutant DnaAcos protein, which causes overinitiation in vivo, is insensitive to the inactivating factor, suggesting a requirement for this negative control in vivo. We propose that a soluble factor controls initiation through down-regulation of DnaA protein.

Cell cycle control: prokaryotic solutions to eukaryotic problems?

Regulation of the eukaryotic cell cycle involves calcium- and lipid-stimulated kinases acting on cytoskeletal structures; there are two principal reasons for supposing that the regulation of the prokaryotic cell cycle may be fundamentally the same. First, evidence for their fundamental difference is still missing and, second, evidence for prokaryotic homologues of eukaryotic cell cycle proteins is accumulating. Such proteins include those involved in calcium regulation, such as calmodulin and calcium-dependent kinases, and those involved in lipid regulation, such as protein kinase C. Proteins identified as candidates for cytoskeletal elements now include MukB, a putative contractile protein responsible for chromosome segregation, and FtsZ, the key constituent of the "cytokinetic" ring. These similarities allow the application of powerful prokaryotic model systems to one of biology's most profound, complex and urgent problems: the nature of the regulation of the eukaryotic cell cycle.

Genetically altered levels of inorganic polyphosphate in Escherichia coli.

The ppk gene encoding polyphosphate kinase (PPK), the enzyme in Escherichia coli that makes long chains of polyphosphate (polyP) reversibly from ATP, was disrupted by insertion of a kanamycin resistance gene. Expression of the exopolyphosphatase gene (ppx) immediately downstream of ppk in the operon was likewise disrupted. Cells were also transformed with a high-copy-number plasmid bearing ppk. Genetically altered polyP levels were estimated in cell extracts by the PPK conversion of ADP to ATP. PolyP levels (microgram/10(11) cells) near 2.0 were reduced in the ppk(-)-ppx- mutants to 0.16 and increased more than 100-fold (e.g. 220) in cells transformed with multiple copies of ppk. Mutant cells, lacking the long polyP chains, showed a growth lag following dilution of a stationary-phase culture. PolyP-deficient cells exhibit a striking phenotype in their failure to survive in stationary phase and loss of resistance to heat (55 degrees C) and to oxidants (42 mM H2O2). High polyP levels are also associated with reduced survival.

Fluid membranes with acidic domains activate DnaA, the initiator protein of replication in Escherichia coli.

Acidic phospholipids in a fluid phase dissociate ADP or ATP tightly bound to DnaA protein and, in the presence of ATP and DNA, can restore an inactive ADP form to full activity (Sekimizu, K., and Kornberg, A. (1988) J. Biol. Chem. 263, 7131-7135). Further studies of the interactions between DnaA protein and lipids have used two functional assays: 1) release of ADP or ATP from DnaA and 2) DNA replication upon rejuvenation of an inactive ADP-DnaA protein complex. Among a variety of phospholipids tested were pure synthetic compounds and the mixtures from Escherichia coli auxotrophs (fabA), which are unable to synthesize unsaturated fatty acids and can be supplemented with different acyl derivatives. Fatty acid composition was determined by gas-liquid chromatography and membrane fluidity by fluorescence spectroscopy using 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid requirements of DnaA protein were shown to be: 1) phospholipids in a fluid phase (i.e. above the transition temperature), 2) a charged polar head group, 3) a lamellar phase (i.e. hexagonal II structures were inactive), and 4) a certain degree of fluidity imparted by the fatty acids esterified to the glycerol backbone. This conclusion was based on the incorporation of: 1) cholesterol, known to increase the packing of lipids, or 2) a branched fatty acyl derivative, which exhibits a fluidizing effect similar to that of a cis double bond. Both agents demonstrated that membrane fluidity is required for DnaA protein function in vitro, consistent with early studies of chromosome initiation in growing cells.

Replicatively active complexes of DnaA protein and the Escherichia coli chromosomal origin observed in the electron microscope.

DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.

An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon.

A gene, ppx, that encodes a novel exopolyphosphatase of 513 amino acids (58,133 Da) was found downstream of the gene for polyphosphate kinase, ppk. Transcription of the ppx gene depends on the ppk promoters, indicating a polyphosphate (polyP) operon of ppk and ppx. Exopolyphosphatase, purified to homogeneity from overproducing cells, is judged to be a dimer of 58-kDa subunits. Orthophosphate is released processively from the ends of polyP approximately 500 residues long, but chains of approximately 15 residues compete poorly with polyP as substrate; ATP is not a substrate. Mg2+ (1 mM) and a high concentration of K+ (175 mM) support optimal activity.