DNA synthesis during bacterial conjugation. I. Effect of mating on DNA replication in Escherichia coli Hfr

Conjugation in Escherichia coli involves an oriented transfer of DNA from the Hfr to the F^?. We have examined the course of DNA replication in a donor cell while it is transferring its DNA. Using isotopic density shift for estimating replication, we have shown that mating is accompanied by initiation of a new round of DNA replication in Hfr cells. With the onset of F-mediated transfer replication, the normal vegetative replication in the Hfr appears to be suppressed. Experiments with F′ donors indicate that the transfer of the chromosome is necessary for switching off vegetative replication.     

The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.     

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.     

DNA synthesis in Escherichia coli in the presence of cyanide

The addition of cyanide to an exponentially growing culture of Escherichia coli causes an immediate, but spontaneously reversible, inhibition of DNA synthesis. The small amount of DNA which is synthesized appears to be the product of the chromosomal replication fork. However, a substantial number of single-strand interruptions persist in the newly synthesized DNA, so that covalent linkage to the preformed DNA does not occur for many minutes. Since the presence of cyanide causes a shift from DPN to DPNH, it is suggested that the DPN-linked joining activity in these cells is inhibited because of the lowered DPN concentration.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.     

DNA synthesis during bacterial conjugation: II. Is DNA replication in the Hfr obligatory for chromosome transfer ?

F-mediated transfer replication in a thymine-requiring Hfr of Eseherichia coli containing a thermosensitive dna B gene has been investigated. Ordinarily, DNA synthesis in the Hfr accompanies chromosome transfer at the non-permissive temperature. When the donors are, however, deprived of thymine, transfer occurs without any DNA synthesis.     

Symmetric multifork chromosome replication in fast-growing Escherichia coli

Density transfer experiments were performed on Escherichia coli growing exponentially in rich medium. The results rule out asymmetric multifork “rolling circle” replication for the E. coli chromosome replicating in rich medium, and are consistent with symmetric multifork replication, with the reinitiations taking place on the two daughter chromosomes simultaneously.     

Synchronization of Escherichia coli by Zonal Centrifugation

A zonal centrifugation technique that can select the smallest newborn cells in an exponentially growing culture of Escherichia coli B/r is described.     

EFFECTS OF DIVISION-SYNCHRONIZING HYPOXIC AND HYPERTHERMIC SHOCKS UPON TETRAHYMENA RESPIRATION AND INTRACELLULAR ATP CONCENTRATION

The division of Tetrahymena pyriformis GL cells was synchronized with either seven hypoxic or five hyperthermic (heat) shocks. Hyperthermic shocks of 34°C produced no reduction in respiration rate and only a 19% decline in intracellular ATP concentration. Hypoxic shocks of 0.15% ambient oxygen concentration depressed intracellular ATP concentration 50%. It therefore appears that hypoxic shock, but not hyperthermic shock, reverses progress of Tetrahymena toward fission by reducing ATP concentration through a reduction of the rate of oxidative phosphorylation. After the first synchronized division, whether synchronized by intermittent hypoxia or hyperthermia, total respiration rate increased exponentially at the same rate of increase as total respiration rate in an exponentially growing (log phase) Tetrahymena cell culture. Before the first synchronized division, the total respiration rate increased exponentially but more slowly than after completion of the first synchronized division. The pattern of increase of total respiration during division synchronized by either procedure was different than the pattern of increase of total respiration of synchronous cells observed by Zeuthen.     

Bi-Directional Chromosomal Replication in Salmonella typhimurium

Transducing frequencies of phage P22 lysates prepared from Salmonella typhimurium exponential cultures in minimal and nutrient broth media were compared. The assumption is that cells grown in a minimal medium will have one replication fork per replication unit, but cells in nutrient broth will have multiple replication forks; therefore, the frequency of genetic markers near the origin of replication will be higher in the nutrient broth culture. Analysis of transduction showed a gradient of marker frequencies from the highest (the cysG-ilv region) to the lowest (purE-trpB region) in both clockwise and counter clockwise directions. This supports our previous observation that chromosome replication proceeds bidirectionally from the origin between cysG (109 min on S. typhimurium map) and ilv (122 min) to a terminus in purE-trpB region (20 to 53 min). Since this method avoids possible artifacts of other methods, the results are assumed to reflect the sequence of chromosome replication in exponentially growing cells. Evidence for the existence of multiple replication forks in nutrient broth-grown cells was supported by the following: (i) the marker frequency data fitted the assumption of multiple replication fork formation; (ii) residual deoxyribonucleic acid increase after inhibition of protein synthesis to complete a round of chromosome synthesis which was 44% in cells grown in a minimal medium and 82% in those in nutrient broth; (iii) segregation patterns of the (3)H-thymidine-labeled chromosome strands during subsequent growth in non-radioactive medium were studied by autoradiography, and the number of replication points per chromosome per cell was estimated as 5.6 for the nutrient broth culture and 2.5 for the minimal medium culture. These data support a model of symmetrical and bidirectional chromosome replication.     

Specific Action of Sodium Dodecyl Sulfate on the Sex Factor of Escherichia coli K-12 Hfr Strains

A specific action of sodium dodecyl sulfate (SDS) on the sex (F) factor in the integrated state of Escherichia coli K-12 Hfr H strain is reported. Growth of Hfr cells in Penassay Broth containing SDS results in the elimination of part or all of the F factor, yielding low and nonfertile variants of defective Hfr type and F⁺ cells and also F⁻ derivatives. Appearance of such variants was generally observed after the culture reached stationary phase. The frequencies of F⁻ cells then increased. F⁻ cells were usually isolated as the major population among survivors. Some defective variants of Hfr cells with an intermediate fertility between standard Hfr and F⁺ cells had lost sensitivity toward the male-specific ribonucleic acid phage M12. Other defective Hfr variants with as much or less fertility than standard F⁺ cells had also all lost sensitivity to phage M12. On single-colony isolation, they segregated nonfertile female H cells which, when infected with F, could restore high fertility with oriented transfer of the chromosome the same as that of the original Hfr H. Also, sensitivity to phage M12 was regained. Female H cells were characterized as those lacking fertility but still retaining a small segment of F or sfa locus at the original part of the chromosome, where newly infected F could attach. Similar results were obtained with two other Hfr strains. A possible mechanism of the specific action of SDS is discussed.     

Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K.

The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol. It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA. An Hfr strain designated as Hfr(R6K) no. 1 was thus obtained and characterized. It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C. The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol. These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.     

Stability of the carrier state in bacteriophage M13-infected cells.

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.     

Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome

We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density-shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow-cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.     

Chromosome transfer and Hfr formation by F in rec+ and recA strains of Escherichia coli K12.

We attempted to assess the role of Hfr clones in chromosome transfer by F⁺ populations. We thought that any Hfr-independent component of fertility might be affected to a different extent by the recA mutation than was the Hfr component. However, the rate of Hfr formation and the efficiency of chromosome transfer were reduced to an equal extent (× 100-fold) by the recA mutation. Such experiments therefore provide no evidence for an Hfr-independent component. It appeared that Type II strains, which were thought to suffer a defect in Hfr formation, actually produced fertile clones but had a secondary defect which affected the persistence of these clones. Thus, evidence from Type II strains is also not useful for examining the quantitative contribution of Hfr cells to F⁺ transfer.     

oriX: A new replication origin in E. coli

Replication of the chromosome of E. coli at 42°C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42°C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxal mutant of one of these Hfrs, we have shown that replication proceeds at 42°C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

Deoxyribonucleic Acid-Deoxyribonucleic Acid Hybridization Assay for Replication Origin Deoxyribonucleic Acid of Escherichia coli

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.     

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria

A direct method for calculating the average number of replicationforks per chromosome in an exponentially growing bacterial cultureand the fraction of reinitiation after an inductive treatmentof the initiation step is presented. This method has allowedthe development of REPLJCON, a computer program designed forthe resolution of the algorithm and simulation of the bacterialchromosome replication. Using REPLJCON the following parameterscan be obtained: average number of replication forks per chromosome,time required for the complete replication cycle, average amountof DNA per nucleotide, gene frequency of any chromosomal locusand reinitiation fraction. The use of this analysis also permitsthe determination of the uni- or bidirectionality of replication. Received on September 30, 1987; accepted on May 10, 1988     

Replication of the F''lac sex factor in the cell cycle of Escherichia coli

Summary The timing of replication of an F'lac during the cell cycle of Escherichia coli B/r has been investigated at different growth rates to clarify the relationship of F factor replication to cell division and the replication of the bacterial chromosome.Cells of a lacZ — strain carrying an F'lac were separated according to their ages in an exponentially growing population after the culture was pulse labelled with a radioactive precursor of DNA and pulse induced for the synthesis of -galactosidase. The amount of label incorporated at different cell ages reflects the state of replication of the bacteriial chromosome, while the amount of enzyme synthesized in response to a short period of induction is assumed to reflect the state of replication of the F'lac.The F'lac replicates at a time somewhat more than half way through the cell cycle at all growth rates investigated. This time is clearly distinguishable from the time of initiation of chromosomal replication at some of the growth rates studied, implying the existence of at least some different control elements in the replication of these two replicons.The regulation of F'lac replication has been further studied by following F'lac replication in temperature sensitive mutants, which are defective in the initiation of chromosomal replication at elevated temperatures.