Isolation of coliphage lambda ghosts able to adsorb onto bacterial cells.

We have examined three methods of lambda ghost production, starting with the [3H]leucine-labelled phage, purified by CsCl density gradient sedimentation. Ghosts obtained by the osmotic shock or by incubation in 5 M LiCl do not adsorb on bacteria. Ghosts obtained by the treatment with the chelating agent EDTA and purified by CsCl density gradient sedimentation possess well preserved adsorption properties and are virtually free of DNA and infectious phage particles.

Importance of two ATP-binding sites for oligomerization, ATPase activity and chaperone function of mitochondrial Hsp78 protein.

The yeast mitochondrial chaperone Hsp78, a homologue of yeast cytosolic Hsp104 and bacterial ClpB, is required for maintenance of mitochondrial functions under heat stress. Here, Hsp78 was purified to homogeneity and shown to form a homo-hexameric complex, with an apparent molecular mass of approximately 440 kDa, in an ATP-dependent manner. Analysis of its ATPase activity reveals that the observed positive cooperativity effect depends both on Hsp78 and ATP concentration. Site-directed mutagenesis of the two putative Hsp78 nucleotide-binding domains suggest that the first nucleotide-binding domain is responsible for ATP hydrolysis and the second one for protein oligomerization. Studies on the chaperone activity of Hsp78 show that its cooperation with the mitochondrial Hsp70 system, consisting of Ssc1p, Mdj1p and Mge1p, is needed for the efficient reactivation of substrate proteins. These studies also suggest that the oligomerization but not the Hsp78 ATPase activity is essential for its chaperone activity.

Architecture of the Streptomyces lividans DnaA protein-replication origin complexes.

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separated by a spacer (134 bp). The Streptomyces DnaA protein consists, like all other DnaA proteins, of four domains: domain III and the carboxyterminal part (domain IV) are responsible for binding of ATP and DNA, respectively. Binding of the DnaA protein to the entire oriC region analysed by electron microscopy showed that the DnaA protein forms separate complexes at each of the clusters of DnaA boxes, but not at the spacer separating them. In vivo mutational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA boxes allows the DNA-bound DnaA protein to induce bending and looping of the oriC region. As it was shown by electrophoretic mobility shift assay and "one hybrid system", two domains, I and III, facilitate interactions between DnaA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DNA-bound DnaA protein can form dimers. Thus, interactions between DnaA molecules may bring two clusters of DnaA boxes separated by the spacer into functional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes which are not fully developed, or huge nucleoprotein aggregates.

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

A physical map of the bioAB region in the lambda bio transducing phage.

The center of the pBopA promoter-operator region for the bioABFCD operon is located 1.71 kb clockwise from the att lambda site on the Escherichia coli genome, as determined by the position of the p131 (IS1) insertion. The order of several bio endpoints to the right of p131 is lambda bio267, 122, 169, 74, 1, and 69. The endpoints of the two bio deletions, delta 61 in bioA and delta 3h in bioB, were also determined.

Leftward transcription in the Escherichia coli bio operon does not require products of the rightward transcript.

The amount of leftward transcription in the Escherichia coli bio operon, as measured by hybridization and by beta-galactosidase assays in lac-bio fusion strains, was determined in bacteria lysogenic for lambda bio phage carrying different amounts of DNA corresponding to rightward message, and in bacteria with polar or nonpolar bioB mutations. The positions of the bioB endpoints in relation to the pB promoter were determined by electron microscopy of heteroduplexes. Normal rates of leftward transcription were found in all cases, except that the shortest lambda bio (lambda bio showed a 2- to 3-fold increase in leftward transcription, which was not abolished by the presence of a wild-type bio operon in trans. These results indicate that no product of the rightward transcript is needed to turn on leftward transcription.

Bacteriophage and host mutants causing the rolling-circle lambda DNA replication early after infection.

There are two modes of bacteriophage lambda DNA replication during its lytic development in Escherichia coli cells. The circle-to-circle (theta) replication predominates at early stages of the phage growth, whereas rolling-circle (sigma) replication occurs late after infection to produce long concatemers that serve as substrates for packaging of lambda DNA into phage proheads. The mechanism regulating the switch from theta to sigma replication remains unknown. Our previous genetic studies indicated that the bacteriophage lambda Pts1piA66 mutant cannot replicate at 43 degrees C in the wild-type E. coli host, but it can replicate in the dnaA46(ts) mutant. Density shift experiments suggested that the parental DNA molecules of the infecting phage enter sigma replication. Here, using electron microscopy, we demonstrate that as soon as 5 min after infection of the dnaA46(ts) mutant by the lambdaPts1piA66 phage at 43 degrees C, the sigma replication intermediates are highly predominant over theta replication intermediates, contrary to the wild-type conditions (wild-type bacteria infected with the lambdaP(+) phage). The initiation of replication of the lambdaPts1piA66 mutant at 43 degrees C was strongly inhibited in the dnaA(+) host, as demonstrated by electron microscopy and by pulse-labeling of the phage-derived plasmid replicon. Implications for the mechanism of the regulation of the switch from theta to sigma replication mode are discussed.

The presence of two DnaA-binding sequences is required for an efficient interaction of the Escherichia coli DnaA protein with each particular weak DnaA box region.

Using an electron microscopic method for visualizing interactions of the Escherichia coli DnaA protein with weak DnaA-binding DNA sequences, we found that DnaA binds effectively to two separated weak DnaA box regions located on the same DNA fragment. As expected, no DnaA-DNA interactions were detected when both DnaA box regions were mutagenized to the sequence totally incapable of binding DnaA. However, when only one of these two regions was mutagenized, the lack of interactions between DnaA and DNA was observed not only at the scrambled DnaA box but also at the second weak DnaA box region. These results indicate that for the efficient binding of DnaA to a weak DnaA box region, the presence of at least two such DNA sequences is necessary. Our finding also suggests that binding of DnaA protein to weak DnaA box sequences may be cooperative. In addition, we found that DnaA-mediated transcription termination in vivo requires two DnaA boxes, one of them is a weak one. It seems, therefore, that some mechanisms of regulation of transcription and DNA replication by DnaA, that involve interactions of DnaA with weak DnaA boxes, may be more complicated than initially proposed.

A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis.

Electron microscopy is a powerful technique for analysis of DNA replication intermediates. However, isolation of replicating DNA molecules from living cells is tricky and difficult, especially in the case of small DNA molecules (such as bacterial plasmids) whose initiation of replication is not easily synchronized. Here a relatively simple and rapid method for efficient isolation of replicating plasmid molecules from unsynchronized Escherichia coli cultures is described. The efficiency of this procedure is high enough for electron microscopy analysis of plasmid replication intermediates appearing in living cells in normal growth conditions. Under optimal conditions, using standard procedures of isolation of plasmid DNA, it is possible to achieve a content of only as few as 0.02 percent of replication intermediates in a plasmid DNA sample. The described method allowed us to enrich up to 100-fold the fraction of replication intermediates suitable for microscopic analysis among all plasmid molecules.

Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme.

The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector. The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level. Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.

Coliphage lambda ghosts obtained by osmotic shock or LiCl treatment are devoid of J- and H-gene products.

We have proved by acrylamide gel electrophoresis that DNA-free ghosts of bacteriophage lambda obtained by osmotic shock (S-ghosts), or by incubation in 5 M-L-Cl (L-ghosts) do not possess the proteins specified by the genes J and H. Electron microscopy of L-ghosts showed that they are devoid of the whole tail tip, composed of the basal part and the tail fibre. The lack of the J-gene product, which is believed to be the tail fibre, explains why S- and L-ghosts do not adsorb to susceptible bacteria. Our results suggest that the H-gene product, which is modified after translation, is situated in the basal part of the tail.

DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli.

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.

A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species.

The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans. In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes. However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown. Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene. This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication. These defects in diverse cellular processes found in the insertional cgtA mutant of V. harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

Formation of macromolecular complexes and other effects of DNA treatment with diethyl and dimethyl sulfate: physico-chemical and electron microscopic studies.

In vitro exposures of isolated DNA to one of the two carcinogenic and mutagenic chemicals, diethylsulfate or dimethylsulfate, induces several kinds of physicochemical and morphological alterations. These changes are detectable by a variety of independent techniques. A fraction of DNA treated briefly with either of these two chemicals moves during velocity centrifugation experiments less rapidly than the bulk of control DNA and more rapidly through gels during electrophoresis. This apparent decrease in size is paralleled by the formation of large DNA aggregates with mobilities indicating molecular weights several times that of the untreated, control DNA. The presence of a basic protein in the incubation mixture increases the rate of formation of such complexes. the tendency of the alkylated DNA to bind to both biological and non-biological materials is reflected in the increased attachment of DNA to columns built with methyl-esterified serum albumin and in its quantitative retention on nitrocellulose filters. DNA exposed to dimethylsulfate decreases its density in CsCl gradients. A mixture of two or more DNAs of different densities exposed to this chemical produces an u.v.-absorbing band which is found in such gradients at an intermediate density. If the alkylation reaction is carried out in the presence of a protein, a portion of DNA bands at a density intermediate between the density of DNA and that of the protein, even in the presence of an ionic detergent in the gradient. Under the electron microscope the alkylated DNA shows multiple single-strand breaks and peeling-off whiskers of denatured DNA. Aggregates of DNA molecules become visible upon further incubation of DNA with the alkylating agent. We suggest that the DNA-DNA and DNA- protein complexes play an important role in the process of carcinogenesis and mutagenesis.

SeqA, the Escherichia coli origin sequestration protein, is also a specific transcription factor.

The SeqA protein is a negative regulator of initiation of DNA replication in the Escherichia coli chromosome. Here, we demonstrate that SeqA stimulates transcription from the bacteriophage lambda pR promoter both in vivo and in vitro. The activity of the lambda pL promoter was found not to be affected by this protein. SeqA-mediated stimulation of pR was dependent on the state of template methylation: transcription was activated on fully methylated and hemimethylated templates but not on an unmethylated template. Using electrophoretic mobility shift assay and electron microscopy, we demonstrated that SeqA interacts specifically with a pR promoter region located on both fully methylated and hemimethylated DNA molecules, but not on unmethylated DNA. The activity of SeqA was found to affect the initiation of lambda plasmid replication positively in vivo, probably via pR-dependent expression of lambda replication genes and transcriptional activation of ori lambda. We conclude that, apart from its function in the control of DNA replication, SeqA is also a specific transcription factor.

Structure and regulation of the dnaA promoter region in three Streptomyces species.

The regulatory region of the Streptomyces dnaA gene comprises a single promoter and two DnaA boxes that are located upstream of the promoter. Comparative analysis of the dnaA promoter region from S. chrysomallus, S. lividans and S. reticuli revealed that the location, spacing and orientation of the DnaA boxes are conserved. In vitro studies demonstrated that efficient binding of the Streptomyces DnaA protein to DNA requires the presence of two DnaA boxes. In vivo analysis of dnaA promoter mutants deleted for one or both DnaA boxes indicated that the dnaA gene is autoregulated. However, the degree of derepression observed is relatively modest.

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.