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1.  Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli1 
Journal of Bacteriology  1968;96(4):939-949.
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.
PMCID: PMC252402  PMID: 4879568
2.  Influence of Starvation for Methionine and Other Amino Acids on Subsequent Bacterial Deoxyribonucleic Acid Replication 
Journal of Bacteriology  1966;92(3):609-617.
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 (N15, C13) 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.
PMCID: PMC276298  PMID: 5332080
3.  Inititation and termination of chromosome replication in Escherichia coli subjected to amino acid starvation. 
Journal of Bacteriology  1980;142(1):236-242.
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.
PMCID: PMC293937  PMID: 6246063
4.  Control of the Synthesis of Macromolecules During Amino Acid and Thymine Starvation in Bacillus subtilis 
Journal of Bacteriology  1968;95(5):1813-1827.
Studies of Maaløe, 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 3H-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 32P incorporation into teichoic acid is normal. Chloramphenicol prevents quasiosmotic thymineless death and also inhibits 32P incorporation into teichoic acid. Chromosome-synthesizing cultures suffer thymineless death of two types: quasiosmotic death, and death insusceptible to osmotic rescue.
PMCID: PMC252216  PMID: 4967776
5.  Sequence of Genes Replicated in Salmonella typhimurium as Examined by Transduction Techniques1 
Journal of Bacteriology  1970;102(2):320-333.
5-Bromouracil (BU) was pulsed into the genome of synchronously growing cells of an F− strain of Salmonella typhimurium of LT2. BU-labeled genes were transduced with P22 phage to a series of recipient auxotrophs. When BU was incorporated early in the replication cycle, the transducing markers that had hybrid densities were those that lie between 9 and 12 o'clock on the genetic map. When BU was incorporated during the terminal period of the synchronous cycle, the transducing particles had hybrid densities for genes that lie from 1 to 8 o'clock clockwise. When phages were prepared on cells in which the middle period was BU-labeled, transducing particles with hybrid densities appeared for genes that lie in two separate regions: between 7 and 9 o'clock and between 12 and 2 o'clock. Analysis of the map sequences of the transduced BU genes, the relative frequency of transduction for each marker, and the time sequence of replication led to a hypothesis that the origin of replication is near the isoleucine-valine gene on the chromosome map. As for direction of replication, several models were considered, including the possibility that replication may proceed in both directions in the same chromosome. It was also found that the aroB, cysG, and strA genes are cotransduced and strA and aroC are also cotransduced. The relative order of the four genes was found to be aroB, cysG, strA, aroC, but the orientation in the circular map was not determined.
PMCID: PMC247554  PMID: 4911538
6.  The Stringent Response and Cell Cycle Arrest in Escherichia coli 
PLoS Genetics  2008;4(12):e1000300.
The bacterial stringent response, triggered by nutritional deprivation, causes an accumulation of the signaling nucleotides pppGpp and ppGpp. We characterize the replication arrest that occurs during the stringent response in Escherichia coli. Wild type cells undergo a RelA-dependent arrest after treatment with serine hydroxamate to contain an integer number of chromosomes and a replication origin-to-terminus ratio of 1. The growth rate prior to starvation determines the number of chromosomes upon arrest. Nucleoids of these cells are decondensed; in the absence of the ability to synthesize ppGpp, nucleoids become highly condensed, similar to that seen after treatment with the translational inhibitor chloramphenicol. After induction of the stringent response, while regions corresponding to the origins of replication segregate, the termini remain colocalized in wild-type cells. In contrast, cells arrested by rifampicin and cephalexin do not show colocalized termini, suggesting that the stringent response arrests chromosome segregation at a specific point. Release from starvation causes rapid nucleoid reorganization, chromosome segregation, and resumption of replication. Arrest of replication and inhibition of colony formation by ppGpp accumulation is relieved in seqA and dam mutants, although other aspects of the stringent response appear to be intact. We propose that DNA methylation and SeqA binding to non-origin loci is necessary to enforce a full stringent arrest, affecting both initiation of replication and chromosome segregation. This is the first indication that bacterial chromosome segregation, whose mechanism is not understood, is a step that may be regulated in response to environmental conditions.
Author Summary
Management of cell growth and division in response to environmental conditions is important for all cells. In bacteria, nutritional downturns are signaled by accumulation of the nucleotide ppGpp. Amino acid starvation causes a programmed change in transcription, known as the “stringent response”; ppGpp also causes an arrest of cell cycle in bacteria, whose mechanism has not been thoroughly investigated. Here, we show that E. coli cells, when the stringent response is in effect, complete chromosomal replication but do not initiate new rounds and arrest with an integer number of chromosomes. The number of chromosomes corresponds to the growth rate prior to arrest. In polyploid arrested cells, the chromosomal regions at which replication initiates are segregated, whereas the termini regions remain colocalized. The E. coli chromosome remains decondensed and unsegregated during arrest and rapidly resumes replication and segregation, concomitant with chromosome condensation, upon release. The protein SeqA, a DNA binding protein and negative regulator of replication, is necessary for enforcing this arrest.
PMCID: PMC2586660  PMID: 19079575
7.  Effect of Thymine Limitation on Chromosomal Deoxyribonucleic Acid Synthesis in Proteus mirabilis 
Journal of Bacteriology  1972;111(3):750-757.
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 μg/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 μg/ml to 0.2 μg/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 μg/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.
PMCID: PMC251349  PMID: 4559826
8.  Coupling Between Chromosome Completion and Cell Division in Escherichia coli 
Journal of Bacteriology  1973;115(3):786-795.
The relationship between termination of chromosome replication and cell division was investigated in Escherichia coli B/r. Synchronous cultures of E. coli B/r growing in glucose minimal medium or subjected to a nutritional shift-up were exposed to chloramphenicol, rifampin, mitomycin C, or nalidixic acid, and the ability of cells to divide in the presence of the inhibitors was measured. It was found that cell division became resistant to inhibitors of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis at approximately the same stage in the division cycle in all situations investigated. When the synchronous glucose-grown cultures were temporarily exposed to chloramphenicol early in the division cycle and then exposed to mitomycin C or nalidixic acid immediately after removal of chloramphenicol, the cells did not divide. In contrast, when DNA synthesis was inhibited by thymine starvation immediately after temporary exposure to chloramphenicol, cells divided. The results suggest that DNA chain elongation is completed in some cells in the absence of protein synthesis, but that additional steps involving specific RNA or protein synthesis, or both, may be required for processing the chromosomal structures to the form which is necessary for division. This processing, which normally occurs concurrent with DNA synthesis and is prevented by inhibitors of DNA synthesis, may trigger division. Alternatively, in the absence of protein synthesis, all aspects of chromosome formation may be completed, but final transcriptional events which are essential for division cannot take place until the complete synthesis of a critical amount of specific proteins.
PMCID: PMC246322  PMID: 4580567
9.  Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes 
Journal of Virology  1978;28(1):53-65.
Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for “normal” DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of “Okazaki pieces,” and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated α-32P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.
PMCID: PMC354247  PMID: 212613
10.  DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli. 
Journal of Bacteriology  1987;169(8):3701-3706.
In synchronized culture of Escherichia coli, the specific arrest of phospholipid synthesis (brought about by glycerol starvation in an appropriate mutant) did not affect the rate of ongoing DNA synthesis but prevented the initiation of new rounds. The initiation block did not depend on cell age at the time of glycerol removal, which could be before, during, or after the doubling in the rate of phospholipid synthesis (DROPS) and as little as 10 min before the expected initiation. We conclude that the initiation of DNA replication is not triggered by the preceding DROPS but requires active phospholipid synthesis. Conversely, when DNA replication initiation was specifically blocked in a synchronized culture of a dnaC(Ts) mutant, two additional DROPS were observed, after which phospholipid synthesis continued at a constant rate for at least 60 min. Similarly, when DNA elongation was blocked by thymine starvation of a synchronized culture, one additional DROPS was observed, followed by linear phospholipid accumulation. Control experiments showed that specific inhibition of cell division by ampicillin, heat shock, or induction of the SOS response did not affect phospholipid synthesis, suggesting that the arrest of DROPS observed was due to the DNA replication block. The data are compatible with models in which the DROPS is triggered by an event associated with replication termination or chromosome segregation.
PMCID: PMC212454  PMID: 3301809
11.  The effect of antibiotic therapy on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. 
The Journal of Hygiene  1975;75(2):275-292.
Chickens in groups of 40 were infected orally with a nalidixic acid-resistant mutant of Salmonella typhimurium and then fed continuously on diets containing ampicillin, chloramphenicol, furazolidone, neomycin, oxytetracycline, polymixin, spectinomycin, streptomycin or a mixture of trimethoprim and sulphadiazine. The amount of S. typhimurium excreted in their faeces was estimated at intervals by culture on brilliant green agar containing sodium nalidixate, both direct and after enrichment in selenite broth; the amount of Escherichia coli excreted was estimated by culture on MacConkey agar. The feeding of diets containing 500 mg./kg. of ampicillin, furazolidone, neomycin, polymixin, spectinomycin or streptomycin or 100 mg./kg. of trimethoprim and 500 mg./kg. of sulphadiazine for 46 days reduced to a varying degree the amount of S. typhimurium and E. coli excreted, the greatest reduction in S. typhimurium being brought about by the last treatment. The effect was less obvious when the concentration of the antibiotics in the food was decreased fivefold. An important reason for the very limited effect of some of the antibiotics was the emergence of antibiotic-resistant populations of S. typhimurium and E. coli. High concentrations of antibiotic-resistant organisms also arose in the faeces of the chickens fed diets containing tetracyclines and chloramphenicol, treatments which had no apparent effect on the amount of S. typhimurium and E. coli excreted. Much of the antibiotic resistance encountered was determined by R factors, a particular R factor usually being found in the E. coli populations of individual chickens before it was found in their S. typhimurium populations. No S. typhimurium or E. coli were isolated that possessed R factors determining resistance to polymixin, furazolidone or trimethoprim. No S. typhimurium or E. coli were isolated that were polymixin-resistant and no S. typhimurium that were furazolidone-resistant. The few trimethoprim-resistant S. typhimurium isolated were thymine-dependent. The feeding of diets containing the higher concentrations of trimethoprim/sulphadiazine, neomycin, furazolidone or ampicillin for 9 days reduced the amount of S. typhimurium excreted. After the withdrawal of these diets, the amount of S. typhimurium excreted increased to the numbers found in chickens given ordinary diets throughout; the chickens that had been given trimethoprim/sulphadiazine or furazolidone did not remain faecal excreters of S. typhimurium longer than the chickens that had been given ordinary diets. Similar results were obtained with trimethoprim/sulphadiazine when the start of the 9-day treatment period was delayed for an extra 9 days or when it was extended to 18 days.
PMCID: PMC2130296  PMID: 1100714
Journal of Bacteriology  1962;83(4):919-923.
Shapira, Jacob (Consolidated Veterans Administration Hospital, Little Rock, Ark.), Lois Lowden, and Ralph Hale. Inhibition of a thymine-deficient mutant of Escherichia coli by 5-substituted uracils. J. Bacteriol. 83:919–923. 1962.—Small inocula of well-washed cells of a thymine-requiring mutant of Escherichia coli were incubated in a thymine-containing glucose-salts medium with a variety of 5-substituted pyrimidines and pyrimidine ribosides. After a lag phase, the turbidity of the cultures increased appreciably which, in the case of 5-ethyluracil and 5-ethyluridine, was primarily due to an elongation of the cells. 5-Ethyluracil at low thymine concentrations increased the lag phase and decreased the rate and final amount of growth. At high thymine concentrations, it had less effect on the final turbidity of the cultures. The inhibition index for this compound was relatively constant, suggesting competitive inhibition.
Several other pyrimidine analogues inhibited growth. The nucleosides of 5-bromouracil and 5-aminouracil were no more effective than the free bases. The ribosides of 5-ethyluracil and 5-butyluracil were appreciably more inhibitory than the free bases and were the most potent compounds tested. It is likely that the inhibition of growth is a reflection of the effect of these compounds on ribonucleic acid synthesis by the cells.
PMCID: PMC279375  PMID: 13911280
13.  Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes 
Journal of Virology  1971;8(4):455-468.
A temperature-inducible mutant of temperate Bacillus bacteriophage φ105 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 φ105 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.
PMCID: PMC376219  PMID: 5002012
14.  Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine1 
Journal of Bacteriology  1971;106(1):197-203.
Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 μg/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 μg/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 μg/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 μg/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 μg/ml and 0.2 μg/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 μg/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.
PMCID: PMC248662  PMID: 4929692
15.  Bi-Directional Chromosomal Replication in Salmonella typhimurium 
Journal of Bacteriology  1973;115(1):168-176.
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 3H-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.
PMCID: PMC246227  PMID: 4577740
16.  Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo. 
Molecular and Cellular Biology  1988;8(5):1923-1931.
A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.
PMCID: PMC363370  PMID: 3386630
17.  Correlation between size and age at different events in the cell division cycle of Escherichia coli. 
Journal of Bacteriology  1980;143(3):1241-1252.
The variability of (i) the B period between birth and initiation of chromosome replication, (ii) the U period between initiation of chromosome replication and initiation of cell constriction, and (iii) the interdivision period (tau) have been estimated for slowly growing Escherichia coli B/r F. Cultures synchronized by the membrane elution technique were pulse-labeled with [3H]thymidine or continuously labeled with [3H]thymine. After fixation, the pattern of deoxyribonucleic acid replication was analyzed by electron microscopic radioautography. Cell length was found to increase exponentially with age at two different slow growth rates. The coefficient of variation of the B period was estimated to be 60%, that of the U period was 29%, and that of the interdivision period was 12%. From these values and the coefficient of variation of length at different cell cycle events were calculated a negative correlation between the B and U period (r = -0.9) and a positive correlation between length at birth and cell separation (r = 0.6). Initiation of chromosome replication and cell constriction were strictly correlated both with respect to age (r = 0.7) and length (r = 0.8). On the other hand, length at initiation of chromosome replication was distantly correlated with age (r = 0.1) or length at birth (r = 0.3). This low correlation excludes a model in which chromosome initiation is controlled by a random event in the B period. It favors a model in which chromosome initiation occurs at a particular distributed size independent of cell division.
PMCID: PMC294487  PMID: 6997267
18.  Utilization of 5-Bromouracil by Thymineless Bacteria 
Journal of Bacteriology  1967;93(1):86-89.
Several thymineless Escherichia coli strains have been examined for their ability to replicate their deoxyribonucleic acid when bromouracil is substituted for thymine. The procedure we describe was used to identify a thymineless strain with characteristics relatively favorable to its use in bromouracil labeling experiments. In addition, mutants with an “absolute” thymine requirement could be easily distinguished from one with a “leaky” thymine requirement.
PMCID: PMC314972  PMID: 5335905
19.  Insensitivity of Chromosome I and the Cell Cycle to Blockage of Replication and Segregation of Vibrio cholerae Chromosome II 
mBio  2012;3(3):e00067-12.
Vibrio cholerae has two chromosomes (chrI and chrII) whose replication and segregation are under different genetic controls. The region covering the replication origin of chrI resembles that of the Escherichia coli chromosome, and both origins are under control of the highly conserved initiator, DnaA. The origin region of chrII resembles that of plasmids that have iterated initiator-binding sites (iterons) and is under control of the chrII-specific initiator, RctB. Both chrI and chrII encode chromosome-specific orthologs of plasmid partitioning proteins, ParA and ParB. Here, we have interfered with chrII replication, segregation, or both, using extra copies of sites that titrate RctB or ParB. Under these conditions, replication and segregation of chrI remain unaffected for at least 1 cell cycle. In this respect, chrI behaves similarly to the E. coli chromosome when plasmid maintenance is disturbed in the same cell. Apparently, no checkpoint exists to block cell division before the crippled chromosome is lost by a failure to replicate or to segregate. Whether blocking chrI replication can affect chrII replication remains to be tested.
Chromosome replication, chromosome segregation, and cell division are the three main events of the cell cycle. They occur in an orderly fashion once per cell cycle. How the sequence of events is controlled is only beginning to be answered in bacteria. The finding of bacteria that possess more than one chromosome raises the important question: how are different chromosomes coordinated in their replication and segregation? It appears that in the evolution of the two-chromosome genome of V. cholerae, either the secondary chromosome adapted to the main chromosome to ensure its maintenance or it is maintained independently, as are bacterial plasmids. An understanding of chromosome coordination is expected to bear on the evolutionary process of chromosome acquisition and on the efficacy of possible strategies for selective elimination of a pathogen by targeting a specific chromosome.
PMCID: PMC3350373  PMID: 22570276
20.  Rates of Gyrase Supercoiling and Transcription Elongation Control Supercoil Density in a Bacterial Chromosome 
PLoS Genetics  2012;8(8):e1002845.
Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound “nucleoid.” The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σD) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σC) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σC+σD. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb “supercoil sensor” inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (−) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σD at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.
Author Summary
A 9-kb module called the “supercoil sensor” was used to measure supercoil density at 10 positions in the 4.8-Mb Salmonella Typhimurium chromosome. The sensor includes a Lac operon flanked by a pair of directly repeated DNA–binding sites for the γδ recombinase. Measurements of chromosomal supercoil levels and the RNA polymerase elongation rates were made at various positions within the 6 potential macrodomains of the chromosome. Transcription and gyrase catalytic rates were mechanistically linked. Gyrase mutants with impaired activity caused the loss of from 30% to >95% of the diffusible supercoiling throughout most of the chromosome, while treatment with rifampicin that temporarily blocked transcription restored most of the lost supercoils in gyrase mutants. A gyrase defect also caused transcription elongation rates to decrease across the chromosome, and a mutation that reduced RNA polymerase efficiency increased average chromosome supercoiling levels. A model in which topoisomerases act close to highly transcribed operons to equilibrate the supercoil flux generated by transcription suggests that matched rates of gyrase turnover and transcription elongation speed determine the average supercoil density in bacterial chromosomes.
PMCID: PMC3420936  PMID: 22916023
21.  Stalled replication fork repair and misrepair during thymineless death in Escherichia coli 
Starvation for DNA precursor dTTP, known as ‘thymineless death’ (TLD), kills bacterial and eukaryotic cells alike. Despite numerous investigations, toxic mechanisms behind TLD remain unknown, although wrong nucleotide incorporation with subsequent excision dominates the explanations. We show that kinetics of TLD in Escherichia coli is not affected by mutations in DNA repair, ruling out excision after massive misincorporation as the cause of TLD. We found that the rate of DNA synthesis in thymine-starved cells decreases exponentially, indicating replication fork stalling. Processing of stalled replication forks by recombinational repair is known to fragment the chromosome, and we detect significant chromosomal fragmentation during TLD. Moreover, we report that, out of major recombinational repair functions, only inactivation of recF and recO relieves TLD, identifying the poisoning mechanism. Inactivation of recJ and rep has slight effect, while the recA, recBC, ruvABC, recG and uvrD mutations all accelerate TLD, identifying the protection mechanisms. Our epistatic analysis argues for two distinct pathways protecting against TLD: RecABCD/Ruv repairs the double-strand breaks, whereas UvrD counteracts RecAFO-catalyzed toxic single-strand gap processing.
PMCID: PMC3965187  PMID: 20465561
22.  More Precise Mapping of the Replication Origin in Escherichia coli K-12 
Journal of Bacteriology  1974;120(1):1-5.
The origin of replication in Escherichia coli K-12 was mapped by determining the rate of marker replication during a synchronous round of replication. Four isogenic strains were made lysogenic for λind− and for phage Mu-1, with Mu-1 integrated into a different chromosomal location in each strain. Cultures were starved for amino acids to allow completion of chromosome replication cycles and then starved for thymine in the presence of amino acids, and a synchronous cycle of replication was initiated by the addition of thymine. Samples were exposed to radioactive thymidine at intervals, deoxyribonucleic acid was extracted, and the rate of marker replication was determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization to filters containing Mu-1, λ, and E. coli deoxyribonucleic acid. The results confirm that the origin of replication is near ilv. The travel times of the replication forks, calculated from the data obtained for cultures with doubling times of approximately 40 and 61 min, are 40 and 52 min, respectively.
PMCID: PMC245722  PMID: 4607435
23.  Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure. 
Molecular and Cellular Biology  1995;15(6):2942-2954.
Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.
PMCID: PMC230525  PMID: 7760792
24.  Salmonella enterica Serovar Typhimurium RamA, Intracellular Oxidative Stress Response, and Bacterial Virulence  
Infection and Immunity  2004;72(2):996-1003.
Escherichia coli and Salmonella enterica serovar Typhimurium have evolved genetic systems, such as the soxR/S and marA regulons, to detoxify reactive oxygen species, like superoxide, which are formed as by-products of metabolism. Superoxide also serves as a microbicidal effector mechanism of the host's phagocytes. Here, we investigate whether regulatory genes other than soxR/S and marA are active in response to oxidative stress in Salmonella and may function as virulence determinants. We identified a bacterial gene, which was designated ramA (342 bp) and mapped at 13.1 min on the Salmonella chromosome, that, when overexpressed on a plasmid in E. coli or Salmonella, confers a pleiotropic phenotype characterized by increased resistance to the redox-cycling agent menadione and to multiple unrelated antibiotics. The ramA gene is present in Salmonella serovars but is absent in E. coli. The gene product displays 37 to 52% homology to the transcriptional activators soxR/S and marA and 80 to 100% identity to a multidrug resistance gene in Klebsiella pneumoniae and Salmonella enterica serovar Paratyphi A. Although a ramA soxR/S double null mutant is highly susceptible to intracellular superoxide generated by menadione and displays decreased Mn-superoxide dismutase activity, intracellular survival of this mutant within macrophage-like RAW 264.7 cells and in vivo replication in the spleens in Ityr mice are not affected. We concluded that despite its role in the protective response of the bacteria to oxidative stress in vitro, the newly identified ramA gene, together with soxR/S, does not play a role in initial replication of Salmonella in the organs of mice.
PMCID: PMC321585  PMID: 14742546
25.  Role of RecA and the SOS Response in Thymineless Death in Escherichia coli 
PLoS Genetics  2010;6(3):e1000865.
Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA–damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS–inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.
Author Summary
A long-standing enigma in the fields of DNA repair and cancer chemotherapy is why it is that cells starved of the base thymine die rapidly. This process, called thymineless death (TLD), is conserved in bacterial, yeast, and human cells and is the mode of action of important cancer chemotherapeutic drugs. Tumors that become resistant to those drugs have ceased to die from TLD. Despite its ubiquity, importance, and having been studied for more than 50 years, the mechanism(s) of TLD remained elusive. Here we show that a large fraction of TLD requires RecA, the central protein in homologous recombinational (HR) DNA repair, and activation of the bacterial DNA–damage (or SOS) response, which RecA controls. We find that of the 40 or so proteins upregulated during an SOS response, SulA, an inhibitor of cell division, accounts for most of how SOS–activation causes TLD. In cells undergoing TLD, we observe blocked replication of the E. coli chromosome followed by loss of DNA near the replication origin then terminus. This implies that much of TLD results from an irreversible cell-cycle checkpoint that blocks cell division when single-stranded DNA (the SOS–inducing signal) accumulates and that the rest results from DNA destruction, models for which are presented.
PMCID: PMC2832678  PMID: 20221259

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