Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli

The effect of p-fluorophenylalanine (FPA) on deoxyribonucleic acid (DNA) synthesis and chromosome replication was studied in a thymine-requiring mutant of Escherichia coli. The rate and extent of chromosome replication were followed by labeling the DNA with isotopic thymine and a density marker, bromouracil. The DNA was extracted and analyzed by CsCl gradient centrifugation. The block in chromosome replication caused by high concentrations of FPA occurred at the same point on the chromosome as that caused by amino acid starvation. In a random culture, DNA in cells treated with FPA replicated only slightly slower than the DNA from cells that were not exposed to the analogue. In cultures which had been previously starved for thymine, however, the DNA from the cells treated with FPA showed a marked decrease in the rate and extent of replication. It was concluded that the E. coli cell is most sensitive to FPA when a new cycle of chromosome replication is being initiated at the beginning of the chromosome.     

Transport of Donor Deoxyribonucleic Acid into the Cell Interior of Thymine-starved Bacillus subtilis with Chromosomes Arrested at the Terminus

The chromosomes of a tryptophan(-), thymine(-) double auxotroph of Bacillus subtilis were uniformly aligned at the chromosome terminus by an amino acid starvation treatment. By subsequent incubations, the starved culture was rendered competent, while its state of synchronous chromosome arrest was maintained by thymine starvation. The competent, chromosome-arrested cells were transformed for three unlinked markers, located in two different chromosome regions. Shortly after addition of deoxyribonucleic acid, the cell walls were removed with lysozyme in a medium containing deoxyribonuclease and no thymine, and the protoplasted culture was assayed for single and double transformants. It was found that markers both near and distant from the terminus entered freely into the cell interior. There was no important difference in the relative frequency of entry of different markers between synchronously arrested cells and nonsynchronized control cultures. It is concluded that entry of a given marker into the cell interior can occur even if the replication site of the chromosome is stationary at a location distant from the locus of the resident homolog of the entering marker. A mechanism of donor deoxyribonucleic acid entry involving homology at the replication fork is excluded.     

Genetic Mapping in Bacillus subtilis by 5-Bromouracil Sensitization to Ultraviolet Inactivation of Transforming Activities

A new method for chromosome mapping of Bacillus subtilis Marburg is presented which is based on the sensitization to ultraviolet irradiation of transforming deoxyribonucleic acid that has incorporated 5-bromouracil instead of thymine. Deoxyribonucleic acid was extracted at intervals from the outgrowing spores of a thymine-requiring mutant incubated with 5-bromodeoxyuridine and subjected to a definite dose of ultraviolet irradiation. The residual activities of various genetic markers were assayed by transformation. The marker activity of deoxyribonucleic acid that had incorporated 5-bromodeoxyuridine was approximately 10 times as sensitive to ultraviolet irradiation as that of normal deoxyribonucleic acid. The markers proximal to the replication origin were sensitized at earlier times of outgrowth than distal markers. The chromosome replication in outgrowing spores was sufficiently synchronous and allowed the definite determination of when a marker became sensitized by incorporation of 5-bromodeoxyuridine. The time, designated "sensitization time," was estimated by plotting the logarithmic values of relative residual activities versus incubation times. The map constructed with sensitization times as a measurement showed good agreement with those constructed by other methods. The replication of the chromosome under the described conditions appeared to occur in the following marker order: (purA, hisA)-(purB)-(thr, pyrA)-(metC)-(leuA)-(lys, trpC, metB).     

Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication

Billen, Daniel (University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Tex.), and Roger Hewitt. Influence of starvation for methionine and other amino acids on subsequent bacterial deoxyribonucleic acid replication. J. Bacteriol. 92:609-617. 1966.-A study has been made of the subsequent replicative fate of deoxyribonucleic acid (DNA) synthesized during amino acid starvation by several multiauxotrophic strains of Escherichia coli. Using radioisotopic and density labels and a procedure whereby total cellular DNA is analyzed, we have confirmed and extended a recent report that the DNA made during amino acid starvation behaves anomalously during subsequent DNA replication. When 5-bromouracil (BU) serves as the density lable, 40% or more of the DNA synthesized during starvation will subsequently fail to replicate during three cell generations. Selective amino acid effects were noted. In two methionine-requiring bacteria, methionine deprivation appeared to be of singular importance in influencing the subsequent replicative fate of the DNA made in its absence.When a non-BU density label (N(15), C(13)) was utilized, the effects of amino acid starvation were less obvious. Although the DNA synthesized during complete amino acid starvation in a methionine-requiring E. coli was subsequently more slowly replicated, most of the DNA was finally duplicated during three generations of growth. If methionine was present during starvation for other required amino acids, the subsequent replication rate of the DNA synthesized during this time was more nearly normal, and complete replication was observed. The results have been interpreted as indicating that DNA synthesized during amino acid starvation, and especially during methionine starvation, is somehow altered, and that BU substitution for thymine may interfere with the restoration of such DNA to its replicative state.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: origin of chromosome replication during exponential growth.

We have investigated the behavior, during exponential growth, of strains of Escherichia coli carrying a dnaA(Ts) mutation that has been suppressed by the integration of the F-like R plasmid R100.1. We present evidence showing that replication in these strains proceeds largely from the normal chromosome origin at 30 degrees C, a permissive temperature for the dnaA(Ts) gene product, whereas, at 42 degrees C, replication proceeds largely from the integrated plasmid. These conclusions are based on measurements made by deoxyribonucleic acid:deoxyribonucleic acid hybridization of the relative frequencies of the prophages Mu-1 and lambdaind- and R100.1 integrated at known locations on the E. coli chromosome in these Hfr strains.     

Inititation and termination of chromosome replication in Escherichia coli subjected to amino acid starvation.

Initiation and termination of chromosome replication in an Escherichia coli auxotroph subjected to amino acid starvation were examined by following the incorporation of [3H]thymidine into the EcoRI restriction fragments of the chromosome. The pattern of incorporation observed upon restoration of the amino acid showed that starvation blocks the process of initiation prior to deoxyribonucleic acid synthesis within any significant portion of the EcoRI fragment which contains the origin of replication, oriC. In this experiment, no incorporation of [3H]thymidine into EcoRI fragments from the terminus of replication was observed, nor was it found when a dnaC initiation mutant was used to prevent incorporation at the origin which might have obscured labeling of terminus fragments. Thus amino acid starvation does not appear to block replication forks shortly before termination of replication. Attempted synchronization of replication initiation by including a period of thymine starvation subsequent to the amino acid starvation led to simultaneous incorporation of [3H]-thymidine into all EcoRI fragments within the 240-kilobase region that surrounds oriC. It is shown that the thymine starvation step allowed initiation and a variable, but limited, amount of replication to occur.     

Effect of Thymine Limitation on Chromosomal Deoxyribonucleic Acid Synthesis in Proteus mirabilis

The effects of thymine limitation on the rates of growth, deoxyribonucleic acid (DNA) synthesis, and increase in viable cell number for a thymine auxotroph of Proteus mirabilis were investigated. At thymine concentrations of 1.0 mug/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 mug/ml to 0.2 mug/ml, mass synthesis continued at the preshift rate for several hours. In contrast, the rate of DNA synthesis immediately decreased, resulting in a decrease in the DNA to mass ratio to about one-half of its normal level. Viable counts remained constant for several hours after the reduction in thymine concentration, and enlarged cells and multicellular "snakes" were formed. The rate of DNA synthesis was reduced at thymine concentrations below approximately 1.7 mug/ml. The addition of thymine to cultures which had been completely starved for thymine increased the rate of DNA synthesis to at least twice its normal value; this suggests that extra rounds of chromosome replication can be induced in P. mirabilis as previously observed in Escherichia coli.     

Initiation of deoxyribonucleic acid replication in Escherichia coli B: uncoupling from mass/deoxyribonucleic acid ratio.

In Escherichia coli growing at different rates, the ratio of cell mass to the number of chromosome origins tended to be constant at the time of the initiation of deoxyribonucleic acid (DNA) replication. This observation led to the assumption that the initiation event is controlled in some way by cell mass, e.g., by a growth-dependent synthesis of an initiator or dilution of a repressor. We have now found that the initiation of DNA synthesis can be uncoupled from cell mass. We used a synchronous culture of newly divided cells of E. coli B which was obtained by the membrane elution technique (C.E. Helmstetter, J. Mol. Biol. 24: 417-427, 1967) and was starved for an amino acid. Upon restoration of the amino acid, the cells not only divided at a size that was smaller than normal, but also initiated DNA replication long before they could increase their masses to reach the expected ratio of mass/DNA presumably required for initiation.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Initiation of Deoxyribonucleic Acid Synthesis After Thymine Starvation of Bacillus subtilis

Evidence for premature initiation of deoxyribonucleic acid (DNA) replication after thymine starvation of Bacillus subtilis W23T(-) is presented, based on (i) increase in the number of ade(+) relative to met(+) transformants yielded by the DNA isolated from cultures after starvation (the ade(-) marker being near the origin of replication, whereas met(-) is close to the terminus), and (ii) increase in both the initial rate and final level of tritiated thymine incorporation in the presence of chloramphenicol after release from starvation. The marker ratio data agree quantitatively with the hypothesis that the initiation is induced only on one arm of each chromosome which was replicating prior to starvation.     

Rate stimulation of deoxyribonucleic acid synthesis after inhibition.

The degree to which the rate of deoxyribonucleic acid synthesis in thy- cultures of Escherichia coli is stimulated after a period of thymine starvation is shown to be a function of the concentration of thymine present as well as of the culture doubling time. Inhibition of deoxyribonucleic acid synthesis by nalidixic acid yields comparable results. Periods of thymine starvation exceeding one doubling time appear to cause an irreversible inactivation of a fraction of the replication forks in the culture.     

Effect of Inhibitors of Ribonucleic Acid and Protein Synthesis on the Cyclic Adenosine Monophosphate Stimulation of Plasmid ColE1 Replication

Addition of cyclic adenosine 3'-5'-monophosphate (c-AMP) to growing Escherichia coli cells, colicinogenic for the plasmid ColE1, results in a fourfold stimulation in the rate of synthesis of the plasmid deoxyribonucleic acid (DNA). The stimulation is transient (30 min) and is succeeded by a brief period (30 min) of cessation of plasmid DNA replication. The stimulation of ColE1 DNA replication also occurs in chloramphenicol-treated cells. Rifampin inhibits ColE1 DNA replication in the presence or absence of c-AMP. Employing thymine starvation conditions to stop ColE1 DNA synthesis, it was found that c-AMP, added during the period of thymine starvation, effected a stimulation in the amount of subsequent replication which took place when replicating conditions were restored. The stimulatory effect of c-AMP under these conditions was not prevented by chloramphenicol but was completely eliminated when rifampin was present. Under these conditions, when rifampin was added after the effect of c-AMP was allowed to occur, subsequent replication of the plasmid could take place, but only one round of replication occurred. A model to account for the c-AMP effects is presented.     

Effects of Inhibition and Restoration of Protein Synthesis on the Replication of the R Factor Rts1 in Proteus mirabilis

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.     

Control of the Synthesis of Macromolecules During Amino Acid and Thymine Starvation in Bacillus subtilis

Studies of Maal?e, Lark, and others with amino acid- and thymine-starved cultures revealed successive steps in the biosynthesis of Escherichia coli chromosomes. In this study, the corresponding mechanisms in Bacillus subtilis 168 were examined. Using a strain requiring both thymine and tryptophan, we found that, 3 hr after the start of amino acid starvation, when the deoxyribonucleic acid (DNA) content of the culture had increased 40 to 50%, DNA synthesis ceased. After 4 to 5 hr, 100% of the cells were immune to thymineless death; their chromosomes had presumably been completed. Immune cultures slowly incorporated (3)H-thymine. Thymine incorporation increased 20-fold 30 min after readdition of amino acids, indicating reinitiation of chromosome synthesis. Simultaneous presence of amino acids and thymine was required for reinitiation. If 5-bromouracil (5-BU) was added instead of thymine, newly replicated DNA segments could be separated by centrifugation in CsCl. Analysis of the CsCl fractions by a transformation assay showed that the order in which the markers were synthesized was ade-16, thr-5, leu-8, metB5. Less than half the chromosomes started resynthesis synchronously in 5-BU. Nevertheless, chromosome alignment in the amino acid-starved culture is probably very good: marker frequency analysis of its DNA gives the same normalized frequencies as DNA from "perfectly" aligned spores. Full viability is maintained in the chromosome-arrested culture for 10 hr in thymine-free medium in the absence or presence of amino acids. In the latter condition, protein synthesis proceeds, and the cells filament and become more lysozyme-sensitive. Such cells must be incubated and plated on hypertonic or on slow-growth media; otherwise, they undergo "quasiosmotic" thymineless death. This death is thus apparently not directly attributable to any damage of chromosomal DNA. Further, weakening of the teichoic acid portion of the cell wall is not involved, since (32)P incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits (32)P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue.     

Chromosome Replication in Salmonella typhimurium

The replication of the Salmonella typhimurium chromosome was studied. As with E. coli 15T(-), replication was sequential. After amino acid starvation, replication proceeded from a unique and heritable region of the chromosome. 5-Bromouracil, when substituted for thymine, did not disturb the sequence of replication nor did it initiate extra replication cycles. By labeling the origin and the terminus of the chromosome with (3)H- and (14)C-thymine, respectively, it was possible to determine that the rate of chain elongation decreases as the growth rate decreases. No gap in the replication cycle could be observed.     

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.     

Origin and sequence of chromosome replication in Escherichia coli

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

Thymineless Death in Escherichia coli: Deoxyribonucleic Acid Replication and the Immune State

Thymineless death (TLD) and nalidixic acid (NA) inactivation were studied in multiple auxotrophic strains of Escherichia coli B and B/r. As expected, it was found that both E. coli B and B/r exhibited an "immune state," i.e., a fraction of the population survived inactivation to both TLD and NA. With glucose as a carbon source in minimal medium, 0.1 to 0.3% of strain B and 0.2 to 0.5% of strain B/r survived inactivation; with acetate as the carbon source, the surviving fractions were increased to 1 to 2% and 5 to 7%, respectively. These immune fractions could be increased in magnitude by preincubation in minimal media containing thymine. Systematic analysis of the particular supplements necessary for the immune state indicated that the absence of the required amino acids was essential for the maximal expression of immunity. However, immunity was not abolished in acetate medium even in the presence of the required supplements. Further studies on the replication of deoxyribonucleic acid (DNA) during preincubation indicated that the degree of immunity did not necessarily correlate with the completion of a round of DNA replication. This finding was supported by examining the immune state in synchronous populations. In both glucose and acetate medium, there was no significant change in the degree of immunity to inactivation within the cell cycles of E. coli B and B/r. We concluded that some other event, possibly inhibition of protein synthesis, was necessary in determining the degree of the immune state. DNA replication was investigated after TLD and NA inactivation, and, as expected, it was found that both events led to premature initiation of replication. The only differences observed in the effects of these two processes on DNA synthesis were the following. (i) NA-induced replication was less sensitive to chloramphenicol than was TLD. (ii) TLD-induced replication was unaffected by pretreatment of the cells with mitomycin C, but this pretreatment prevented the replication of DNA after NA treatment. It was suggested that the mechanism of action of NA could involve a monofunctional attack on the DNA.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.