The bacterial chromosome and its manner of replication as seen by autoradiography.
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Autoradiography↗
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[Participation of plasmid F in the replication of the chromosome of an Hfr strain of Escherichia coli K-12].
In the region of plasmid F DNA with coordinates 52,2-55,8 kb, the chr ("chromosome replication") locus has been revealed. A failure in the functioning of this locus in the integrated plasmid, which leads to a temperature-sensitive disturbance in chromosome replication of the Hfr strain and to the changes in its sensitivity to some membranotropic agents. Integration of an F segment containing the chr+ allele into the chromosome of an F-like derivative of such Hfr strain (retaining a mutant part of the F DNA), results in formation of temperature-resistant clones. In these clones, chromosomal replication is controlled by the plasmid replicon at the elevated temperature. It has been concluded that the F plasmid can control chromosome replication of the dna+ HfrC strain of Escherichia coli K-12 and that the product of the chr gene is a membrane protein involved in chromosomal replication.
Chromosome Mapping↗
Size variations and correlation of different cell cycle events in slow-growing Escherichia coli.
Cell lengths have been determined at which cycle events occur in the slow-growing Escherichia coli B/r substrains A, K, and F26. The radioautographic and electron microscope analyses allowed determination of the variations in length at birth, initiation and termination of DNA replication, and initiation of the constriction process and of cell separation. In all three substrains the standard deviation increased between cell birth and initiation of DNA replication. From there on, the standard deviation remained relatively constant until cell separation. These observations are consistent with the presence of a deterministic phase during the cell cycle in which the cell sizes at initation of DNA replication and at cell division are correlated.
Cell Cycle↗
Growth of bacteriophage Mu in Escherichia coli dnaA mutants.
In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.
Bacteriophage mu↗
Cell cycle-specific replication of Escherichia coli minichromosomes.
The timing of Escherichia coli minichromosome replication in the cell division cycle was examined using an improved procedure for studying plasmid replication frequency. Cultures growing exponentially in glucose/Casamino acids minimal medium were pulse-labeled with [3H]thymidine, and the radioactivity incorporated into plasmid DNA in cells of different ages was analyzed. At the end of the labeling period the bacteria were bound to the surface of a nitrocellulose membrane filter, and the radioactivity in new daughter cells, which eluted continuously from the membrane, was quantitated following agarose gel electrophoresis. The minichromosomes replicated during a discrete interval in the cell division cycle that appeared to coincide with initiation of chromosome replication. In contrast, plasmid pBR322 replicated throughout the division cycle at a rate that increased gradually as a function of cell age. The difference in minichromosome and pBR322 replication was clearly discernible in cells harboring both plasmids. It was also found that the 16 kD gene adjacent to oriC was not a determinant of the timing of minichromosome replication during the division cycle. The results are consistent with the conclusion that minichromosome replication frequency is governed by the same mechanism that controls chromosome replication.
Cell Cycle↗
DNA synthesis and genophore replication in Escherichia coli K-12 synchronized with thymine starvation.
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Cell Division↗
Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K-12. Initiation in the absence of oriC.
In stable DNA replication (sdrA/rnh) mutants of Escherichia coli, initiation of rounds of DNA replication occurs in the absence of the normal origin of replication, oriC. To determine whether or not the initiation occurs at a fixed site(s) on the chromosome in sdrA mutants, the DNA from exponentially growing sdrA mutant cells with or without the oriC site (delta oriC) was analyzed for the relative copy numbers of various genes along the chromosome. The results suggest that there are at least four fixed sites or regions of the sdrA delta oriC chromosome from which DNA replication can be initiated in the absence of the oriC sequence.
Chromosome Mapping↗
Orientation of genes in the Bacillus subtilis chromosome.
The orientation of 96 genes on the Bacillus subtilis chromosome was deduced by the analysis of published data. Of these genes, 91 were found to be oriented so that their promoters were proximal to the chromosomal replication origin and their transcription termini to the replication terminus. Transcription of these genes would therefore be co-directional with replication. This chromosomal organization is consistent with the hypothesis advanced for Escherichia coli that bacteria avoid head-on collisions between RNA polymerase and DNA replication proteins by the appropriate orientation of their transcription units.
Bacillus subtilis↗
Congression of unlinked markers and genetic mapping in the transformation of Bacillus subtilis 168.
A thorough examination of cotransformation of two unlinked genetic markers in Bacillus subtilis 168 shows that the two recombinational events do not occur randomly. The cotransformation frequency is dependent on the distance between the two markers as well as on the order in which they replicate in the competent cell. These results indicate that uptake and/or integration of DNA molecules bearing these genetic markers is enhanced at the time these markers replicate in the competent cell.
Bacillus subtilis↗
Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.
Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.
Amino Acids↗
Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli.
DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E. coli cells.
Adenosine Diphosphate↗
The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves.
We have developed a system for the simultaneous labelling of two specific chromosomal sites using two different fluorescent ParB/parS systems. Using this, we demonstrate that the two chromosome arms are spatially arranged in newborn cells such that markers on the left arm of the chromosome lie in one half of the cell and markers on the right arm of the chromosome lie in the opposite half. This is achieved by reorganizing the chromosome arms of the two nucleoids in pre-division cells relative to the cell quarters. The spatial reorganization of the chromosome arms ensures that the two replication forks remain in opposite halves of the cell during replication. The relative orientation of the two reorganized nucleoids in pre-division cells is not random. Approximately 80% of dividing cells have their nucleoids oriented in a tandem configuration.
Cell Division↗
Cell division after inhibition of chromosome replication in Escherichia coli.
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Cell Division↗
Pseudomonas chromosomal replication origins: a bacterial class distinct from Escherichia coli-type origins.
The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida. These origins comprise a second class of bacterial origins distinct from enteric-type origins: both origins function in both Pseudomonas species, and neither functions in Escherichia coli; enteric origins do not function in either pseudomonad. Both cloned sequences hybridize to chromosomal fragments that show properties expected of replication origins. These origin plasmids are highly unstable, are present at low copy number, and show mutual incompatibility properties. DNA sequence analysis shows that both origins contain several 9-base-pair (bp) E. coli DnaA protein binding sites; four of these are conserved in position and orientation, two of which resemble the R1 and R4 sites of the E. coli origin. Conserved 13-bp direct repeats adjacent to the analogous R1 site are also found. No GATC sites are in the P. aeruginosa origin and only four are in the P. putida origin; no other 4-bp sequence is present in high abundance. Both origins are found between sequences similar to the E. coli and Bacillus subtilis dnaA, dnaN, rpmH, and rnpA genes, a gene organization identical to that for B. subtilis and unlike that of E. coli. A second autonomously replicating sequence was obtained from P. aeruginosa that has some properties of bacterial origins.
Base Sequence↗
Initiation of chromosomal DNA synthesis in vitro.
An in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dna A-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.
Chromosomes, Bacterial↗
Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.
The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.
Bacterial Proteins↗
Ordered and sequential binding of DnaA protein to oriC, the chromosomal origin of Escherichia coli.
DnaA protein of Escherichia coli acts in initiation of chromosomal DNA replication by binding specific sequences, termed DnaA boxes in the chromosomal origin, oriC. On binding, it induces a localized unwinding to create a structure recognized by other replication proteins that act subsequently in the initiation process. In this report, we examined the binding of DnaA protein to each of the DnaA boxes in oriC. By gel mobility shift assays, DnaA protein formed at least six discrete complexes. ATP or ADP included in the reaction mixture prior to electrophoresis was required. Chemical cleavage of isolated complexes with 1,10-phenanthroline-copper revealed that DnaA protein binds in an ordered manner to the DnaA boxes in oriC. Preferential binding to one DnaA box (R4) was confirmed by demonstration that a DNA fragment containing it was bound with greater affinity than another DnaA box sequence (R1). In vitro replication activity correlated with a complex formed at a ratio of 30 DnaA monomers/oriC in which all DnaA boxes are occupied. The last site bound is DnaA box R3. This event may be critical in promoting initiation from oriC as it correlates with in vivo observations that binding of DnaA protein to box R3 occurs at the time of initiation of chromosomal replication, whereas other DnaA boxes are bound by DnaA protein throughout the cell cycle (Cassler, M. R., Grimwade, J. E., and Leonard, A. C.(1995) EMBO J. 14, 5833-5841).
Adenosine Diphosphate↗
Dependence of cell division on the completion of chromosome replication in Caulobacter.
The relationship between chromosome replication and cell division in the stalked bacterium Caulobacter crescentus has been investigated. Two compounds, hydroxyurea and mitomycin C, were found to inhibit completely deoxyribonucleic acid (DNA) synthesis while allowing continued cell growth and elongation. When these inhibitors were added to exponentially growing cultures, cell division stopped after 38 min when hydroxyurea was used and after 33 min when mitomycin C was used. The period of continued cell division corresponds closely to the period previously determined for the postsynthetic gap (G2) in the DNA cycle of this organism. These results indicate that cell division is coupled to the completion of chromosome replication in C. crescentus.
Adenine↗