Kinetics of minichromosome replication in Escherichia coli B/r.

Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed.     

A comparison of UV-induction in exponentially growing and restingEscherichia coli B/r Hcr+

Using the method of two separate UV exposures the increase of UV resistance after various induction fluences in growing and resting Escherichia coli B/r Hcr+ was followed. In resting cells, the optimum induction energy fluence was found to be 30 J/m2. In exponentially growing cells testing of induction has proved to be possible only under conditions of postincubation of cells with chloramphenicol after the second fluence. Under these conditions the induction energy fluence up to the observed 50 J/m2 resulted in an increased survival.     

Chromosome replication and cell division in plasmid-containing Escherichia coli B/r.

The kinetics of chromosome replication and cell division have been examined in recA mutants of Escherichia coli B/r containing F' plasmids of various sizes. Plasmid-mediated alterations in growth properties were detected only with the presence of the larger F' plasmids, and were reflected in decreased mean cell sizes and growth rates. The lengths of C and D in all plasmid-containing strains were in accord with the values for plasmid-free parental strains growing with similar generations times. The findings were consistent with an absence of competition between the chromosomal and extrachromosomal replicons for rate-limiting components involved in the initiation of deoxyribonucleic acid synthesis or in the elongation of deoxyribonucleic acid chains.     

Evidence that F'' lac replicates asynchronously during the cell cycle of Escherichia coli B/r.

Summary The replication of F lac was studied in exponentially growing cultures of E. coli B/r. The cells were pulse induced for the synthesis of -galactosidase and their DNA pulse labelled with ³H thymidine. The cells were then separated into age classes by centrifugation through a sucrose gradient in a zonal rotor. Plasmid replication was measured in each age fraction by three methods: the rate at which -galactosidase could be induced, the amount of label incorporated into CCC plasmid DNA which had been separated from chromosomal DNA on agarose gels, and the amount of label incorporated into plasmid DNA which had been separated from chromosomal DNA by ultracentrifugation through CsCl-EtBr gradients. All these methods gave the same result, that replication of F lac occurs in cells of all ages and is not confined to a part of the cell cycle.     

Deoxyribonucleic acid synthesis after inhibition of initiation of rounds of replication in Escherichia coli B/r.

The theory describing the effect of inhibition of initiation of rounds of deoxyribonucleic acid (DNA) replication on the accumulation of DNA is derived, and an analysis is presented which allows the determination of the time C taken to replicate the bacterial chromosome from the kinetic changes in the accumulation of DNA. This analysis is applied to experiments in which inhibition of initiation was achieved by inhibiting protein or protein and ribonucleic acid synthesis with chloramphenicol or rifampin. The results for both antibiotics are identical and indicate that there is a delay of 6 to 11 min in the effect of the antibiotics on initiation of rounds of replication. If this delay is taken into account, then the value of the C period estimated from such experiments agrees with values obtained by other methods, whereas by conventional data evaluation of such experiments the C period would be overestimated. In the low thymine-requiring derivative of Escherichia coli B/r ATCC 12407 used here, the C period was found to be between 38 and 41 min for cultures growing with a mass doubling time of 29 min in glucose-amino acids medium, supplemented with 20 micrograms of thymine/ml.     

Rifampicin and chloramphenicol effects on DNA replication in thymine-prestarved Escherichia coli B/r WP2 thy trp.

When cultures of Escherichia coli B/r WP2 thy trp were prestarved for thymine for 30 min, DNA replication after readdition of thymine was limited to an increase of about 100% in the presence of rifampicin, an antibiotic which inhibits DNA-dependent RNA polymerase. However, chloramphenicol, an antibiotic which blocks protein but not RNA synthesis, did not limit replication. After prolonged thymine prestarvation (55 min) DNA increased only about 50% in the presence of rifampicin, but no such limitation occurred in the presence of chloramphenicol. The ability of a high concentration of rifampicin to limit DNA replication was eliminated by addition of either high or low concentrations of chloramphenicol, indicating that stoichiometric interaction of the antibiotics is not responsible for this effect.     

UV-inducible repair: influence on survival, dimer excision, DNA replication and breakdown in Escherichia coli B/r Her+ cells.

Summary Using a model of double-UV-irradiation with inducing¹ (non-lethal) and lethal fluences² we have studied involvement of UV-inducible functions in post-UV-irradiation restoration processes and survival of Escherichia coli B/r thy^-thy^- Hcr⁺. Cells irradiated with both inducing and lethal fluences differed from cells irradiated with lethal fluence in the following respects: They were more UV resistant; they did not die during postincubation with chloramphenicol³; they exhibited a significant reduction in dimer excision; they were able to resume DNA replication and produce normal-sized DNA molecules in the presence of chloramphenicol. Since induction was provoked in cell prestarved for amino acids it was not associated with damage to points active in replication. However, the inducible product was more important for repair of replicating than non-replicating cells. The data indicate that protein necessary for resumption of DNA synthesis after UV is not constitutive but inducible.Abbreviations ¹IF inducing fluence - ²IF lethal fluence - ³CAP chloramphenicol     

Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases.

Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one. Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes. At the other central positions, uracil was eliminated with different initial velocities. Single stranded phi X 174 DNA fragments were used to study the influence of the sequence. Cytosine bases were deaminated to give uracil by bisulfite treatment. It was shown that the initial excision velocity of two vicinal uracil residues was decreased. The same observation was made for two uracils separated by one base. A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.     

Sensitivity to ultraviolet radiation as a function of DNA content in Escherichia coli B/r.

Populations of Escherichia coli B/r A were grown to log phase at various growth rates determined by the richness of the medium. The genome content, G, was calculated from log phase doubling times by means of the Cooper-Helmstetter formula. Cell volumes were measured and found to vary linearly with this genome content. Cells with various DNA contents were prepared for ultraviolet irradiation and plated for dark repair under similar conditions. The resulting logarithmic survival curves were all similar in shape: convex up, with straight line portions having approximately the same slope (D0 = 11.4 +/- 0.2 J/m2). The shoulders however increase in width with calculated DNA content giving an extrapolation number which varies roughly as exp(G) or exp (0.6 Gmax).     

Initiation of chromosome replication in dnaA and dnaC mutants of Escherichia coli B/r F.

Regulatory aspects of chromosome replication were investigated in dnaA5 and dnaC2 mutants of the Escherichia coli B/r F. When cultures growing at 25 degrees C were shifted to 41 degrees C for extended periods and then returned to 25 degrees C, the subsequent synchronous initiations of chromosome replication were spaced at fixed intervals. When chloramphenicol was added coincident with the temperature downshift, the extend of chromosome replication in the dnaA mutant was greater than that in the dnaC mutant, but the time intervals between initiations were the same in both mutants. Furthermore, the time interval between the first two initiation events was unaffected by alterations in the rate of rifampin-sensitive RNA synthesis or cell mass increase. In the dnaC2 mutant, the capacities for both initiations were achieved in the absence of extensive DNA replication at 25 degrees C as long as protein synthesis was permitted, but the cells did not progress toward the second initiation at 25 degrees C when both protein synthesis and DNA replication were prevented. Cells of the dnaA5 mutant did not achieve the capacity for the second initiation event in the absence of extensive chromosome replication, although delayed initiation may have taken place. A plausible hypothesis to explain the data is that the minimum interval is determined by the time required for formation of a supercoiled, membrane-attached structure in the vicinity of oriC which is required for initiation of DNA synthesis.     

Amino acid sequence of the L-arabinose-binding protein from Escherichia coli B/r.

The amino acid sequence of the L-arabinose-binding protein of Escherichia coli B/r was determined by sequenator analyses of reduced and S-pyridylethylated L-arabinose-binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage by cyanogen bromide. BNPS-skatole, hydroxylamine, mild acid hydrolysis, limited trypsin digestion, chymotrypsin subdigestion, and subdigestion with Staphylococcus aureus protease V8. The COOH-terminal sequence was determined using bovine carboxypeptidases A and B and amino acid analyses. The L-arabinose-binding protein was determined to contain 306 amino acid residues, the sequence of which is presented below.     

Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r.

The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.     

Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract.

The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract.     

A comparison of DNA content between two substrains of Escherichia coli B/r.

The average DNA content per cell was measured in steady-state cultures of two substrains of $\text{E. coli}\text{B}\text{r}$ growing at various rates at 37°C. The DNA content of substrain $\text{B}\text{r}$ F was consistently lower than that of substrain $\text{B}\text{r}$ A. It is suggested that the differences in DNA contents are consequences of strain-specific differences in the relationship between chromosome replication and the division cycle of $\text{E. coli.}$     

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.