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PubMed2004

Multicopy plasmids affect replisome positioning in Bacillus subtilis.

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

Bacillus subtilis↗
PubMed1968

Timing of enzyme synthesis during outgrowth of spores of Bacillus cereus. II. Relationship between ordered enzyme synthesis and deoxyribonucleic acid replication.

Experiments were carried out to determine whether, during outgrowth of bacterial spores, deoxyribonucleic acid (DNA) replication provided the basis by which ordered transcription was controlled. During outgrowth, significant DNA synthesis does not occur until just prior to the onset of cell division. However, incorporation of radioactive DNA precursors into DNA is observed within 5 to 10 min after the initiation of germination. By employing a thymine-requiring auxotroph and (3)H-bromodeoxyuridine, this incorporation appears to be a result of DNA replication and not repair synthesis. For the following reasons it was concluded that, during outgrowth, transcriptional processes were not ordered by DNA replication. (i) In a thymine auxotroph, thymine addition did not alter the periodicity of induced alpha-glucosidase and histidase synthesis during outgrowth. (ii) DNA synthesis was inhibited 80% by 5-fluoro-2'-deoxyuridine (FUdR), and, after a 5-min lag, completely by mitomycin C, but these inhibitors exerted a differential effect on induced histidase synthesis. Enzyme synthesis was insensitive to FUdR but was inhibited by mitomycin C, presumably as a result of cross-linking of the complementary DNA strands.

Adenine↗
PubMed2004

Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA.

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.

Bacterial Proteins↗
PubMed2001

The replication-recombination connection: insights from genomics.

One amazing characteristic of DNA processing enzymes is their breakneck speed. However, considering the metabolic traffic on DNA, it is not surprising that accidents that interrupt the replication process and require immediate repair occur. Recombination is an example of a repair mechanism, and as a result of accidents, the arrested or broken fork becomes the recombination substrate. Repair proteins are on hand to assist at accident sites, but responses may be sensitive to conditions and depend on the organism. Genomics has been used to identify genetic clues that indicate the occurrence of accidents and to detect sites of recombination. DNA and protein features that may affect replication and recombination are examined and discussed.

Bacteria↗
PubMed2001

Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity.

We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non-permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double-strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.

Base Sequence↗
PubMed1997

Mechanism of anucleate cell production in the oriC-deleted mutants of Bacillus subtilis.

We constructed oriC-deleted mutants of Bacillus subtilis by integrating the minimal replication region of plasmid pLS32 into the proA (115 degrees), spoIIIJ (360 degrees) and thrS (256 degrees) loci of the chromosome, respectively. All three mutants produced anucleate cells and the DNA/protein ratio was lower than that of the wild-type strain when grown in nutrient broth. However, when grown in minimal-glucose medium, the frequency of anucleate cells was reduced in all of them and the DNA/protein ratio was restored to normal. Especially, the oriC-deleted mutant in which the plasmid was integrated near oriC produced almost no anucleate cell. These results indicate that initiation frequency of chromosome replication from the integrated plasmid origin were reduced disproportionately to cell mass increase in rich medium, which in turn disrupted coordination between DNA replication cycle and cell division cycle. The locations of the plasmid origin relative to the natural oriC locus affected the production of anucleate cell remarkably, suggesting that partition mechanism of chromosome was also impaired by the translocation of its replication origin.

Bacillus subtilis↗
PubMed2005

Compartmentalization of prokaryotic DNA replication.

It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

Bacillus subtilis↗
PubMed2001

Bacterial conjugation: running rings around DNA.

Helicases are active in many aspects of DNA replication, recombination, repair and transcription. An integral membrane bacterial protein assembly involved in the transfer of DNA between cells has been shown to resemble a ring helicase, suggesting that it hydrolyzes ATP to pump DNA through a central channel.

Conjugation, Genetic↗
PubMed2006

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus.

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.

Caulobacter crescentus↗
PubMed1977

Terminus region of the chromosome in Escherichia coli inhibits replication forks.

Induction of prophage P2sig5 at 42 degrees caused replication of the bacterial chromosome in a dnaA mutant of Escherichia coli. The P2sig5 is integrated in this strain near the metG locus, which is at min 47 on the genetic map. The regions of the chromosome replicated after prophage induction have been determined by means of DNA-DNA hybridization with various DNAs obtained from Proteus mirabilis/E. coli F' merogenotes and from lambda specialized transducing phage. The replication was initiated at the prophage site and was bidirectional. Most of the replication occurred in a counterclockwise direction on the genetic map, and the replication quickly proceeded to the aroD locus (min 37). The replication forks were retarded between aroD and rac (min 31) loci, although the rac locus was finally replicated. A more severe inhibition of replication occurred between the rac and trp (min 27) loci. It is proposed that the replication terminus is near the rac locus and that the terminus inhibits replication forks.

Chromosomes, Bacterial↗
PubMed2000

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation.

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.

Bacterial Outer Membrane Proteins↗
PubMed1984

The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression.

For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity. The trfA gene is transcribed as part of a polycistronic operon which also includes kilD. Transcription of this operon is negatively regulated by the products of the trfB/korD/korA and korB loci. Mini replicons previously studied in detail lack the korB locus and have copy numbers significantly higher than RK2 itself. Here we report that korB in trans expresses incompatibility towards RK2 replicons either when the korB gene dosage is high or when it is expressed from a strong foreign promoter. This incompatibility can be largely overcome if a trfA gene which is expressed from a foreign promoter, and is therefore not regulated by korB, is supplied in trans. When korB is introduced in cis to mini RK2 replicons the copy number is reduced to within the range estimated for parental RK2. Deletions in the oriV RK2 region which otherwise cause quite large increases in plasmid copy number have only a small effect when korB is present in cis. These results suggest that korB in combination with trfB may be the overriding copy number control element in RK2 reducing trfA expression to levels limiting for replication.

Bacterial Proteins↗
PubMed1997

Missense mutations in the 3' end of the Escherichia coli dnaG gene do not abolish primase activity but do confer the chromosome-segregation-defective (par) phenotype.

Isogenic dnaG strains of Escherichia coli with the parB and dnaG2903 alleles in the MG1655 chromosomal background displayed the classic par phenotype at the nonpermissive temperature of 42 degrees C. These strains synthesized DNA at 42 degrees C, but remained chromosome segregation defective as determined by cytology. A strain with the dnaG2903 allele was tested for its ability to support DNA replication of a primase-dependent G4ori(c)-containing M13 phage derivative by quantitative competitive PCR (QC-PCR). The dnaG2903 strain converted the single-stranded DNA into double-stranded replicative form DNA at 42 degrees C. These results indicate that DnaG2903 retains primase activity at the restrictive temperature. Nucleoids remained unsegregated in the central region of cell filaments at 42 degrees C. The observed suppression of cell filamentation in dnaG sfiA or dnaG lexA double mutants suggests that the SOS response is induced at the restrictive temperature in parB and dnaG2903 strains but fails to account entirely for the cell filamentation phenotype. ParB and DnaG2903 presumably can synthesize primer RNA for DNA replication, but may be defective in their interactions with DNA replication proteins, cell cycle regulatory factors, or the chromosome segregation apparatus itself.

Alleles↗