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PubMed2002

Differential effects of Kid toxin on two modes of replication of lambdoid plasmids suggest that this toxin acts before, but not after, the assembly of the replication complex.

Kid is a small protein that is encoded by plasmid R1. It is a toxin that belongs to a killer system that ensures the stability of the plasmid in host cells. The results of previous studies have suggested that Kid is an inhibitor of DNA replication, possibly acting at the onset of initiation. Here, the authors tested the effects of Kid on orilambda-intitiated and oriJ-initiated replication, which may be driven by both the newly assembled replication complex and the heritable complex. It was found that Kid inhibits only replication that is driven by the newly assembled replication complex. The authors also report that Kid inhibits ColE1-like plasmid replication in vivo, in agreement with the previously reported inhibition of ColE1 during in vitro replication. It is proposed that the Kid toxin acts at the level of replication either by preventing de novo assembly of the replication complex or by impairing the functional interactions of the replication complex at the initiation stage.

Bacterial Proteins↗
PubMed2001

Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Agrobacterium tumefaciens↗
PubMed1978

Chromosome replication in an Escherichia coli dnaA mutant integratively suppressed by prophage P2.

Escherichia coli CRT4624-P2sig5 is a dnaA mutant in which integration of the prophage P2sig5 has occurred at the attP2II site (min 85). This strain was integratively suppressed, and when cells were shifted to 42 degrees C replication was initiated at a site in or near the P2 prophage. Initially, this replication occurred primarily in the direction that corresponds to the clockwise direction on the genetic map. Replication also occurred in the counterclockwise direction, but the initiation of replication in this direction occurred approximately 40 min later than the initiation of replication in the other direction. Because of this delay, the replication forks that traveled in the clockwise direction were the first to arrive in the region of the replication terminus. These replication forks ceased replication near the aroD locus (min 37), and it is proposed that the replication terminus is between the aroD and rac loci (min 31). A model is proposed for the cycle of chromosome replication in this strain at 42 degrees C.

Chromosomes, Bacterial↗
PubMed1979

Nucleotide sequence of the origin of replication of the Escherichia coli K-12 chromosome.

The origin of replication, oriC, of the Escherichia coli chromosome was mapped within a DNA segment of 422 base pairs. The nucleotide sequence of this segment was determined. The source of DNA for the sequence analysis was a minichromosome constructed in vivo, consisting exclusively of chromosomal DNA and a minichromosome constructed by cloning in vitro. The nucleotide sequence of the replication origin is characterized by a high degree of repetitiveness due to both inverted and direct repeats. Sequence homologies were found between portions of the replication origins of E. coli and phages lambda and G4. This suggests similarities in some steps in the initiation of replication of the different replicons.

Base Sequence↗
PubMed1998

Effects of purified SeqA protein on oriC-dependent DNA replication in vitro.

In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.

Bacterial Outer Membrane Proteins↗
PubMed1998

Characterization of dnaC2 and dnaC28 mutants by flow cytometry.

Escherichia coli strains containing thermosensitive dnaC alleles were studied by flow cytometry. Strains containing either the dnaC2 or dnaC28 allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of dnaC mutants for cell cycle studies is discussed.

Bacterial Proteins↗
PubMed1998

In vitro replication of mitochondrial plasmid mp1 from the higher plant Chenopodium album (L.): a remnant of bacterial rolling circle and conjugative plasmids?

According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.

Base Sequence↗
PubMed1980

Isolation and characterization of a composite plasmid Rms201 mutant temperature sensitive for replication.

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37 degrees C. When Escherichia coli ML1410(Rms201ts14)(+) was grown at temperatures between 40 and 42 degrees C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)(+) in L-broth was shifted to 42 from 30 degrees C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42 degrees C. At 30 degrees C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42 degrees C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42 degrees C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42 degrees C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 x 10(6) and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 x 10(6). These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.

Ampicillin↗
PubMed2006

Competition of bacteriophage polypeptides with native replicase proteins for binding to the DNA sliding clamp reveals a novel mechanism for DNA replication arrest in Staphylococcus aureus.

Bacteriophages have evolved specific mechanisms that redirect bacterial metabolic pathways to the bacteriophage reproduction cycle. In this study, we characterized the bactericidal mechanism of two polypeptides from bacteriophages Twort and G1 that target the DNA sliding clamp of Staphylococcus aureus. The DNA sliding clamp, which tethers DNA polymerase to its template and thereby confers processivity upon the enzyme, was found to be essential for the viability of S. aureus. Expression of polypeptides TwortORF168 and G1ORF240 in S. aureus selectively inhibited DNA replication which in turn resulted in cell death. Both polypeptides specifically inhibited the S. aureus DNA replicase that was reconstituted in vitro but not the corresponding replicase of Streptococcus pyogenes. We demonstrated that inhibition of DNA synthesis is multifaceted and occurs via binding the DNA sliding clamp: TwortORF168 and G1ORF240 bound tightly to the DNA sliding clamp and prevented both its loading onto DNA and its interaction with DNA polymerase C. These results elucidate the impact of bacteriophage polypeptide expression upon DNA replication in the growing cell.

Amino Acid Sequence↗
PubMed2002

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli.

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.

Bacterial Proteins↗
PubMed2003

Gene duplication with displacement and rearrangement: origin of the bacterial replication protein PriB from the single-stranded DNA-binding protein Ssb.

PriB is a proteobacterial protein that is involved in the pre-primosomal step of DNA replication and, unexpectedly, is encoded with a ribosomal protein operon. Detailed sequence comparisons and analysis of operon organization show that PriB evolved from the single-stranded DNA-binding (Ssb) via gene duplication with subsequent rapid sequence diversification. Duplication of the SSB gene was accompanied by a genome rearrangement which resulted in one of the paralogs retaining the original position, whereas the other was relocated. The functional specialization of the resulting paralogs apparently proceeded in an unexpected fashion: the original Ssb function remained with the relocated paralog, whereas the one within the ribosomal protein operon acquired a new, specialized function in replication.

Amino Acid Sequence↗
PubMed1988

DNA replication.

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Bacterial Proteins↗