Thymine Starvation Research Articles

Use of Thymine Limitation and Thymine Starvation To Study Bacterial Physiology and Cytology

A variety of very basic questions asked 30 to 40 years ago remain unanswered today. How do bacteria select and maintain a defined size, length, and width? How do they control the timing of chromosome replication? What is the mechanism by which they segregate their replicated (or replicating)

Gyrase inhibitors and thymine starvation disrupt the normal pattern of plasmid RK2 localization in Escherichia coli.

Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.

Bacterial programmed cell death systems as targets for antibiotics

Growing experimental evidence has revealed the existence of programmed cell death (PCD) systems in bacteria. Among these is the mazEF system, which is a regulable suicide module located on the chromosome of E. coli and of some other bacteria, including pathogens. Several well-known antibiotics have recently been found to cause cell death in E. coli by indirectly activating this built-in suicide module. These antibiotics belong to two groups: (i) inhibitors of transcription and/or translation; and (ii) inhibitors of folic acid metabolism resulting in thymine starvation. These data, together with the recent elucidation of the crystal structure of mazEF-directed components, hold promise for a rational chemical design of a new class of antibiotics that directly activate chromosomal suicide modules by interacting with their components. Because multi-drug resistance among bacterial pathogens is becoming more widespread, the results obtained might be useful as a basis for producing alternative drugs.

The Escherichia coli mazEF suicide module mediates thymineless death.

In 1954, Cohen and Barner discovered that a thymine auxotrophic (thyA) mutant of Escherichia coli undergoes cell death in response to thymine starvation. This phenomenon, called thymineless death (TLD), has also been found in many other organisms, including prokaryotes and eukaryotes. Though TLD has been studied intensively, its molecular mechanism has not yet been explained. Previously we reported on the E. coli mazEF system, a regulatable chromosomal suicide module that can be triggered by various stress conditions. MazF is a stable toxin, and MazE is an unstable antitoxin. Here, we show that cell death that is mediated by the mazEF module can also be activated by thymine starvation. We found that TLD depends on E. coli mazEF and that under thymine starvation, the activity of the mazEF promoter P(2) is significantly reduced. Our results, which describe thymine starvation as a trigger for a built-in death program, have implications for programmed cell death in both prokaryotes and eukaryotes.

Thymine-starvation up-regulates a region of theArabidopsis thaliana chromosome III containing both the UMP synthase gene and a proximal gene encoding a novel F-box protein

We have cloned theArabidopsis thaliana undine 5′-monophosphate synthase (UMP synthase) locus and characterized transcript levels of this gene as well as the transcript encoding a novel F-box protein found upstream of UMP synthase,Skp1-interacting partner5 (SKIPS). The 31 end of theSKIP5 gene is 405 bp from the 5′ end of the UMP synthase gene. To determine whether these proximate genes are coordinately or independently regulated, we have used an RT-PCR method to quantitate the transcript levels of each gene relative to 18S ribosomal RNA. Previous work has demonstrated an up-regulation of the UMP synthase gene in tobacco callus in the presence of compounds such as 5-fluoroorotate and aminopterin that induce thymine starvation. Here, we present results showing that both the UMP synthase andSKIP5 genes are coordinately up-regulated in the presence of fluoroorotic acid.

RecQ helicases and genome stability: lessons from model organisms and human disease.

Maintaining the integrity of genetic information is fundamental for the life of a cell and the survival of a species. Cells can encounter DNA damage as a consequence of normal cellular metabolism or as a result of exposure to chemical or physical agents. Eukaryotic cells have developed a network of responses in order to deal with DNA damage thereby preserving the integrity of their genetic information. In the presence of extensive genetic insult, a surveillance mechanism or "checkpoint" is activated. The activation of this signal transduction pathway leads to an arrest of cell cycle progression to prevent replication and segregation of damaged DNA molecules and to induce transcription of several repair genes. Existing repair mechanisms are also mobilised, in a coordinated effort to restore the original DNA structure. Genes involved in either cell cycle checkpoints, DNA repair or genes that maintain the fidelity of chromosome segregation are often termed "antimutators" or "caretaker" genes, because they control the stability of the genome and prevent accumulation of mutations in so-called "gatekeeper" genes. This latter group of genes directly regulate the growth of tumours either by inhibiting growth or promoting death. A fundamental requirement for many DNA metabolism processes is the separation of the complementary strands of the DNA duplex. This is promoted by DNA helicases, which unwind nucleic-acid duplexes in an ATP-dependent manner to provide access to the template for proteins of the replication, recombination, repair and transcription machineries. Multiple DNA helicase families have been identified, all containing seven hallmark helicase motifs; members within each helicase family also share sequence homologies beyond and between these motifs. One example is the RecQ helicase family, named after the RecQ protein of Escherichia coli, which was identified during a search for mutants sensitive to thymine starvation. Five members of the RecQ family have been identified in the human genome, and mutations in three of the genes are responsible for genetic diseases that are characterised by genomic instability and a high incidence of cancer. Because mutants in RecQ family genes in other species also have unstable chromosomes, it was proposed that members of the RecQ helicase family play a central role in the maintenance of genomic stability and thereby the prevention of tumorigenesis.

RecQ helicases and genome stability: lessons from model organisms and human disease.

Maintaining the integrity of genetic information is fundamental for the life of a cell and the survival of a species. Cells can encounter DNA damage as a consequence of normal cellular metabolism or as a result of exposure to chemical or physical agents. Eukaryotic cells have developed a network of responses in order to deal with DNA damage thereby preserving the integrity of their genetic information. In the presence of extensive genetic insult, a surveillance mechanism or "checkpoint" is activated. The activation of this signal transduction pathway leads to an arrest of cell cycle progression to prevent replication and segregation of damaged DNA molecules and to induce transcription of several repair genes. Existing repair mechanisms are also mobilised, in a coordinated effort to restore the original DNA structure. Genes involved in either cell cycle checkpoints, DNA repair or genes that maintain the fidelity of chromosome segregation are often termed "antimutators" or "caretaker" genes, because they control the stability of the genome and prevent accumulation of mutations in so-called "gatekeeper" genes. This latter group of genes directly regulate the growth of tumours either by inhibiting growth or promoting death. A fundamental requirement for many DNA metabolism processes is the separation of the complementary strands of the DNA duplex. This is promoted by DNA helicases, which unwind nucleic-acid duplexes in an ATP-dependent manner to provide access to the template for proteins of the replication, recombination, repair and transcription machineries. Multiple DNA helicase families have been identified, all containing seven hallmark helicase motifs; members within each helicase family also share sequence homologies beyond and between these motifs. One example is the RecQ helicase family, named after the RecQ protein of Escherichia coli, which was identified during a search for mutants sensitive to thymine starvation. Five members of the RecQ family have been identified in the human genome, and mutations in three of the genes are responsible for genetic diseases that are characterised by genomic instability and a high incidence of cancer. Because mutants in RecQ family genes in other species also have unstable chromosomes, it was proposed that members of the RecQ helicase family play a central role in the maintenance of genomic stability and thereby the prevention of tumorigenesis.

Effect of mutations for the ruvABC genes on recombination between direct DNA repairs in Escherichia coli strains carrying extended tandem duplication

The formation of haploid and diploid segregants was studied in Escherichia coli strains carrying heterozygous tandem duplications deoA deoB::Tn5/deoC deoD in the deoCABD operon region, in the genome of mutants for ruvABC genes. Homologous recombination in duplications of rec+ strains and in recBC sbcB, recQ and recF mutants, including those with blocks of both the RecBCD and RecF pathway, was shown in our previous work to be similar to adaptive mutagenesis: in this case, practically each cell forms a recombinant on a selective medium. In this work, mutants for ruv genes were found to differ in this respect, forming segregants at a frequency that was decreased by several orders of magnitude. These data confirm the conclusion that the genetic exchange in duplications proceeds through a special pathway of adaptive (or replicative) recombination connected with DNA replication. Upon selection of recombinants under conditions of thymine starvation, recombination cannot also be induced in ruv mutants. The recombinogenic effect of thymine starvation seems to occur at late stages of recombination, which are controlled by ruvABC genes.

Study of RecA-independent homologous recombination and a chromosomal rearrangement in the Escherichia coli strain carrying an extended tandem duplication

A heterozygous tandem duplication in the Escherichia coli deo operon region deoA deoB::Tn5/deoC deoD thr::Tn9 with the total length approximately 150 kb, which was obtained in the conjugational mating in the HfrH strain, was examined. By means of digestion with the NotI enzyme, pulsed-field gel electrophoresis, and the conjugational transfer of the duplication in the F- strain, the chromosomal rearrangement, which occurred in the duplication region upon its stabilization in the bacterial genome, was studied. In a more stable strain, two new NotI sites were shown to appear in the chromosomal region located close to the duplication, which might have resulted from the transposition of the IS50 sequence from Tn5. The data were also obtained indicating the possibility of secondary transposition of the chromosomal segment between the two new NotI sites (approximately 30 kb) in the region located near the duplication. With the use of rec+ and recA strains, two types of haploid and diploid segregants generated by the duplication were studied: DeoD+ (the DeoD+ allele is not expressed in the original duplication due to the polar effect of the deoB::Tn5 insertion) and DeoC DeoD. The segregation of DeoD+ clones was shown to be RecA-dependent, whereas the DeoC DeoD segregants selected on the medium that contained thymine at a low concentration (i.e., under conditions of thymine starvation) appeared at a rather high frequency. However, the relative frequency of haploid clones, which have lost the duplication, strongly decreased in the recA genome among segregants of both types.

Transcription of essential cell division genes is linked to chromosome replication in Escherichia coli

Cell division normally follows the completion of each round of chromosome replication in Escherichia coli. Transcription of the essential cell division genes clustered at the mra region is shown here to depend on continuing chromosomal DNA replication. After chromosome replication was blocked by either nalidixic acid treatment or thymine starvation, the transcription of these cell division genes was repressed significantly. This suggests a way in which cell division is controlled by chromosome replication.

Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli

BackgroundTo maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate.ResultsTo overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate.ConclusionsThis approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.

Essential role for the Legionella pneumophila rep helicase homologue in intracellular infection of mammalian cells.

We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: Involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the φX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'- pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'- pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'- pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the φX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the φX174-type primosome at a single-stranded n'- pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: Involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the φX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the φX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the φX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.

Thymine metabolism and thymineless death in prokaryotes and eukaryotes.

For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation [thymineless death (TLD)]. This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect. Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects. The direct effects involve both single- and double-strand DNA breaks. The former may be repaired effectively, but the latter lead to cell death. DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair. Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process. This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites. Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction. Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features. The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD. There are similarities between TLD of bacteria and death of eukaryotic cells. Also, bacteria have "survival" genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two. A model is presented for a molecular basis of TLD.

Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.

Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication.

Under certain conditions, Escherichia coli cells exhibit either of two altered modes of chromosomal DNA replication. These are inducible stable DNA replication (iSDR), seen in SOS-induced cells, and constitutive stable DNA replication (cSDR), seen in rnhA mutants. Both iSDR and cSDR can continue to occur in the absence of protein synthesis. They are dependent on RecA protein, but do not require DnaA protein or the oriC site. Here we report the requirement for PriA, a protein essential for assembly of the phi X174-type primosome, for both iSDR and cSDR. In priA1(Null)::kan mutant cells, iSDR is not observed after induction by thymine starvation. Replication from one of the origins (oriM1) specific to iSDR is greatly reduced by the priA1::kan mutation. cSDR in rnhA224 mutant cells deficient in RNase HI is also completely abolished by the same priA mutation. In both cases, SDR is restored by introduction of a plasmid carrying a wild-type priA gene. Furthermore, the viability of an rnhA::cat dnaA46 strain is lost at 42 degrees C upon inactivation of the priA gene, indicating the lethal effect of priA inactivation on those cells whose viability depends on cSDR. These results demonstrate that a function of PriA protein is essential for iSDR and cSDR and suggest the involvement of the PriA-dependent phi X174-type primosome in these DnaA/oriC-independent pathways of chromosome replication. Whereas ColE1-type plasmids, known to be independent of DnaA, absolutely require PriA function for replication, DnaA-dependent plasmid replicons such as pSC101, F, R6K, Rts1 and RK2 are able to transform and to be maintained in the priA1::kan strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymine starvation-induced structural changes in Escherichia coli DNA Detection by pulsed field gel electrophoresis and evidence for involvement of homologous recombination

Effect of thymine starvation on Escherichia coli DNA was investigated by using pulsed field gel electrophoresis combined with cell lysis in agarose gel. Post-lysis treatment with restriction enzymes generating relatively large fragments ( NheI, SpeI or XbaI) revealed peculiar electrophoretic profiles specific for thymine-starved cells. Thus a substantial portion of the DNA remained in the origin of electrophoresis (non-migrating DNA), and the amounts of the migrating fragments correspondingly decreased in an inverse relation to the map distance between the origin of replication ( oriC) and each fragment. The formation of non-migrating DNA seems to depend upon the presence of replicated portions of the chromosome (sister duplexes), as judged by the effect ofa preincubation at the non-permissive temperature in a dnaA(Ts) mutant. Electron microscopy showed that the non-migrating fraction of DNA was enriched with such structures as single-stranded tails or gaps and branchings with single-stranded arms. It was also found that the appearance of non-migrating DNA was highly dependent on the functional recA gene and moderately on certain RecF-family genes. These results strongly suggest that homologous recombination between sister duplexes is involved in the formation of the peculiar structures found in non-migrating DNA. A possible causal relationship between the formation of non-migrating DNA and viability loss (thymineless death) is also discussed.

DNA Damage-inducible Replication of the Escherichia coli Chromosome is Initiated at Separable Sites within the Minimal oriC

When Escherichia coli cells are subjected to genetic stress by exposure to agents or conditions that transiently block DNA replication, the mode of DNA replication is profoundly altered. One of the alterations is the induction of inducible stable DNA replication (iSDR) that does not require the initiator protein, DnaA, and occurs despite the presence of rifampin and chloramphenicol, which inhibit the initiation of usual chromosome replication at oriC. It has been demonstrated that iSDR starts primarily from both the oriC and terC regions of the chromosome. To precisely map the iSDR origin (oriM1) located in the oriC region, various oriC fragments were inserted into a plasmid vector derived from pSC101, and the copy number of these plasmid constructs was measured in the presence of rifampin and chloramphenicol after cells were induced for the SOS response by thymine starvation. The results indicated that there are at least two origins for iSDR, within the minimal oriC; one (oriM1A) is located between the BamHI (coordinate +1) and the AvaII(155) sites, and the other (oriM1B) between the AvaII(155) and the Hind III(244) sites. Furthermore, a 263 bp fragment containing oriM1 , which was placed at the attλ site of the chromosome, was found to initiate chromosome replication in the presence of the drugs when cells were starved of thymine. Introduction of additional copies of oriM1 into a cell stimulated initiation of iSDR at oriM1 on the chromosome. The result supported the model that iSDR starts from D-loops created between oriM1 sequences and that the amount of D-loops determines the level of the iSDR activity.