[Bacterial DNA and its replication].
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Bacillus subtilis↗
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Control of plasmid replication in Escherichia coli: correlation of the membrane site of DNA replication with the bacterial segregation unit.
The fate of parental lambda genomes after superinfection of homoimmune lysogenic cells was studied. The data confirm a previous observation that in the presence of the lambda repressor, superinfecting lambda DNA does not become associated with replication sites on the bacterial cell membrane. Under these conditions, the nonreplicating, superinfecting phage genomes do not become associated with the bacterial segregation unit. These results support the concept that the attachment of DNA to the bacterial membrane at specific sites is involved in the control of both chromosome replication and segregation, as predicted by the replicon hypothesis.
Autoradiography↗
Heat induction of prophage phi 105 in Bacillus subtilis: replication of the bacterial and bacteriophage genomes.
A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.
Bacillus subtilis↗
Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding.
Triggering new rounds of chromosomal DNA replication during the bacterial cell cycle is exquisitely regulated, ensuring both proper timing and one round per cycle stringency. A critical first step is stable unwinding of oriC, the chromosomal replication origin, by multiprotein orisome complexes comprising the AAA+ initiator DnaA and modulator proteins that bend DNA. Recently identified oriC-DnaA interactions in Escherichia coli raise important questions regarding the molecular mechanisms that regulate origin unwinding in bacteria. We describe staged binding of E. coli origin recognition proteins and suggest an unwinding switch based on interactions between DnaA-ATP and specialized oriC sites that must be filled during orisome assembly. By focusing multiple regulatory pathways on only a few key oriC DNA-protein interactions, this model includes an efficient way to control unwinding followed by orisome inactivation during the cell cycle. Future studies will determine whether this regulatory scheme is correct and whether it is generally applicable to other bacterial types.
Chromosomes, Bacterial↗
DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes.
A DNA-membrane complex extracted from Bacillus subtilis was studied further as a model system for initiation of bacterial DNA replication in vitro. Of three subcomplexes purified from the crude complex by a combination of CsCl and sucrose gradient centrifugation, the synthetic capability of only one was inhibited significantly by streptovaricin, a known inhibitor of RNA primer formation. A selective enrichment in the level of this subcomplex was obtained by manipulating a thymine-requiring mutant. The synthetic capabilities of an enriched and nonenriched DNA-membrane complex were compared in the presence and absence of streptovaricin. Although the rate and extent of DNA synthesis per unit of protein were approximately the same in the absence of the antibiotic, there was a much greater inhibition of synthesis shown by the enriched complex in the presence of streptovaricin. Although the amount of DNA present in the putative initiation subcomplex was less than 0.3 to 0.4% of the total DNA present in the crude complex, such DNA, except for a few quantitative differences, was still representative of genomic DNA. Newly synthesized DNA hybridized to specific origin- and non-origin-derived restriction fragments of the B. subtilis genome. However, when an elongation inhibitor (ddCTP) was added, hybridization of such DNA to almost all of the nonorigin fragments disappeared or was reduced drastically, whereas origin region hybridization patterns remained strong. The highest level of hybridization in the origin region occurred with a BamHI (B7) restriction fragment of 5.6 kilobases that has been implicated by others as one site initiation in vivo (N. Ogasawara, M. Seiki, and H. Yoshikawa, Nature (London) 281:702-704, 1979; S. J. Seror-Laurent and G. Henckes, Proc. Natl. Acad. Sci. USA 82:3586-3590, 1985).
Bacillus subtilis↗
New targets and strategies for the development of antibacterial agents.
The increasing incidence of bacterial drug-resistance is stimulating the development of strategies targeting previously unexploited mechanisms of antibiotic action. Combinatorial chemistry, which generates molecularly diverse compounds, target-directed strategies, and high-throughput screens are being used to detect potential antibacterial agents. Bacterial DNA replication and cell division are the targets of new screening methods, as are membrane proteins, particularly those constituting efflux pumps; two-component signalling systems are also being targeted. Secondary-screening methods are being developed to find antibiotics that destroy slowly growing or resting bacteria, and to evaluate whether new antibiotics will be active against intracellular bacteria.
Anti-Bacterial Agents↗
Utilization of subsidiary chromosomal replication terminators in Bacillus subtilis.
The Bacillus subtilis merodiploid strain GSY1127 contains a large nontandem duplication of a portion of its chromosome within its left (anticlockwise) replication segment. This causes displacement of the replication terminus region to a noticeably asymmetric location relative to oriC. The utilization of the subsidiary replication terminators, TerIII and TerV, in the merodiploid strain has been compared with that in B. subtilis 168. It is shown that TerIII is utilized to a significant extent in GSY1127 and that TerV is used only marginally at the most. Neither of these terminators is used to a measurable extent in the 168 strain. It is concluded that TerIII and TerV do indeed function as backups to the major terminator TerI, as has been generally thought. It is further concluded that, in the 168 strain, the vast majority of clockwise forks are arrested at the highly efficient TerI terminator, with fork fusion between the approaching forks occurring frequently while the clockwise fork is stationary at TerI.
Bacillus subtilis↗
Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the SpoOJ partitioning protein.
We have developed methods for labelling regions of the Bacillus subtilis chromosome with the nucleotide analogue 5-bromodeoxyuridine (BrdU) and for subcellular visualization of the labelled DNA. Examination of oriC-labelled chromosomes in outgrowing spores has provided direct evidence for active segregation of sister chromosomes. Co-immunodetection of Spo0J and BrdU-labelled DNA has directly confirmed the expected close association between this chromosome partitioning protein and the oriC region of the chromosome. The results provide further support for the notion that bacterial cells use an active mitotic-like mechanism to segregate their chromosomes.
Bacillus subtilis↗
Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I.
A polA12 recA718 double mutant of Escherichia coli, in which DNA polymerase I is temperature sensitive, was unable to maintain normal DNA synthesis or to form colonies on rich media at 42 degrees C. Overproduction of DnaE protein, the polymerizing alpha subunit of DNA polymerase III, restored bacterial DNA replication and cell viability, as well as the PolI-dependent replication of the plasmid carrying dnaE.
DNA Polymerase I↗
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.
Cell Cycle↗
The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex.
Quinolone drugs can inhibit bacterial DNA replication, via interaction with the type II topoisomerase DNA gyrase. Using a DNA template containing a preferred site for quinolone-induced gyrase cleavage, we have demonstrated that the passage of the bacteriophage T7 replication complex is blocked in vitro by the formation of a gyrase-drug-DNA complex. The majority of the polymerase is arrested approximately 10 bp upstream of this preferred site, although other minor sites of blocking have been observed. The ability of mutant gyrase proteins to arrest DNA replication in vitro has been investigated. Gyrase containing mutations in the A subunit at either the active-site tyrosine (Tyr122) or Ser83 (a residue known to be involved in quinolone interaction) failed to halt the progress of the polymerase. A low-level, quinolone-resistant mutation in the B subunit of gyrase showed reduced blocking compared to wild-type. We have demonstrated that DNA cleavage and replication blocking occur on similar time-scales and we conclude that formation of the cleavable complex is a prerequisite for polymerase blocking. Additionally, we have shown that collision of the replication proteins with the gyrase-drug-DNA complex is not sufficient to render this complex irreversible and that further factors must be involved in processing this stalled complex.
Anti-Infective Agents↗
Archaea and the origin(s) of DNA replication proteins.
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Archaea↗
"Replisome"-controlled initiation of DNA replication.
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Bacterial Proteins↗
Origin flexibility governs robust ssDNA engagement by the DnaA initiator.
In model bacteria, initiation of chromosome replication requires engagement of single-stranded DNA by oligomers of the DnaA-family initiator assembled within the origin DNA. Although arrays of double-strand motifs recognized by DnaA are a general feature of the origins, the DnaA-binding single-strand elements are elucidated in only a limited number of species, and the mechanical principles governing their recognition remain elusive. Using the Alphaproteobacterium Caulobacter crescentus, we identify a previously uncharacterized GA-rich single-stranded element in the origin that directly engages DnaA oligomers and is essential for robust initiation. This element is positioned at a subkilobase distance from the DnaA oligomerization region and is brought into proximity through dynamic structural rearrangements. Moreover, DnaA oligomers exhibit an unexpectedly broad yet constrained capacity to accommodate single-stranded sequence variation. These findings provide the molecular basis for origin plasticity, highlighting how origins can diverge while preserving initiation logic.
DNA, Single-Stranded↗
The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication.
The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.
Bacteria↗
[In vitro reaction between methylene blue and the chromosomal DNA of E. coli].
In the present work, the chromosomal DNA isolated from the bacterium E. coli was treated with methylene blue. Formation of a heat-stable complex with methylene blue and E. coli chromosomal DNA, has changed the hyperchromic characteristics of E. coli DNA to some extent. It seemed possible that in vivo binding of the dye with DNA may inhibit the separation of DNA chains thus preventing the DNA replication.
Chromosomes, Bacterial↗
Role of DNA transcription in the initiation of Escherichia coli sex factor (F) DNA replication.
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Bacterial Proteins↗
Role of bacterial chaperones in DNA replication.
Studies on the involvement of chaperone proteins in DNA replication have been limited to a few replication systems, belonging primarily to the prokaryotic world. The insights gained from these studies have substantially contributed to our understanding of the eukaryotic DNA replication process as well. The finding that molecular chaperones can activate some initiation proteins before DNA synthesis has led to the more general suggestion that molecular chaperones can influence the DNA-binding activity of many proteins, including transcriptional factors involved in cell regulatory systems. The DnaK/DnaJ/GrpE molecular chaperone system became a paradigm of our understanding of fundamental processes, such as protein folding, translocation, selective proteolysis and autoregulation of the heat-shock response. Studies on the Clp ATPase family of molecular chaperones will help to define the nature of signals involved in chaperone-dependent proteins' refolding and the degradation of misfolded proteins.
Bacteria↗