Thymine Starvation Research Articles

Environmentally Responsible Bioengineering for Spore Surface Expression of Helicobacter pylori Antigen

The development of genetic technologies and bioengineering are creating an increasing number of genetically engineered microorganisms with new traits for diverse industrial applications such as vaccines, drugs and pollutant degraders. However, the destiny of genetically engineered bacterial spores released into the environment as long-life organisms has remained a big environmental challenge. In this study, an environmentally responsible and sustainable gene technology solution based on the concept of thymine starvation is successfully applied for cloning and expression of a Helicobacter pylori antigen on Bacillus subtilis spore surface. As an example, a recombinant Bacillus subtilis strain A1.13 has been created from a gene fusion of the corresponding N-terminal fragment of spore coat protein CotB in B. subtilis and the entire urease subunit A (UreA) in H. pylori and the fusion showed a high stability of spore surface expression. The outcomes can open the door for developing highly safe spore vectored vaccines against this kind of pathogen and contributing to reduced potential risks of genetically engineered microorganisms released in the environment.

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity.

Thymineless death (TLD) is a rapid loss of viability of unclear mechanism in cultures of thyA mutants starved for thymine/thymidine (T starvation). It is accepted that T starvation repeatedly breaks replication forks, while recombinational repair restores them, but when the resulting futile breakage-repair cycle affects the small replication bubbles at oriC, the origin is degraded, killing the cell. Indeed, cells with increased chromosomal replication complexity (CRC), expressed as an elevated origin/terminus (ori/ter) ratio, die more extensively during TLD. Here we tested this logic by elevating the CRC in Escherichia colithyA mutants before T starvation, anticipating exaggerated TLD. Unexpectedly, TLD remained unaffected by a CRC increase to either the natural limit (ori/ter ratio, ∼6) or the functional limit (ori/ter ratio, ∼16). Moreover, when we forced the CRC over the functional limit (ori/ter ratio, ∼30), TLD lessened. Thus, prior overinitiation does not sensitize cells to TLD. In contradiction with the published results, even blocking new replication initiations by the dnaA(Ts) defect at 42°C fails to prevent TLD. Using the thyA dnaA(Ts) mutant in a new T starvation protocol that excludes new initiations, we show that at 42°C, the same degree of TLD still occurs when chromosomes are demonstrably nonreplicating. Remarkably, 80% of the chromosomal DNA in these nonreplicating T-starved cells is still lost, by an unclear mechanism.IMPORTANCE Thymineless death kills cells of any type and is used in anticancer and antimicrobial treatments. We tested the idea that the more replication forks there are in the chromosome during growth, the more extensive the resulting thymineless death. We varied the number of replication forks in the Escherichia coli chromosome, as measured by the origin-to-terminus ratio, ranging it from the normal 2 to 60, and even completely eliminated replication forks in the nonreplicating chromosomes (ori/ter ratio = 1). Unexpectedly, we found that thymineless death is unaffected by the intensity of replication or by its complete absence; we also found that even nonreplicating chromosomes still disappear during thymine starvation. We conclude that thymineless death can kill E. coli independently of chromosomal replication.

Sources of thymidine and analogs fueling futile damage-repair cycles and ss-gap accumulation during thymine starvation in Escherichia coli

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of action of popular antibacterial and anticancer drugs, yet the mechanisms of TLD are still unclear. TLD comprises three defined phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding key to TLD pathology. We propose that a limited source of endogenous thymine maintains replication forks through the resistance phase. When this source ends, forks undergo futile break-repair cycle during the RED phase, eventually rendering the chromosome non-functional. Two obvious sources of the endogenous thymine are degradation of broken chromosomal DNA and recruitment of thymine from stable RNA. However, mutants that cannot degrade broken chromosomal DNA or lack ribo-thymine, instead of shortening the resistance phase, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal DNA accumulation during the resistance phase is negated during the RED phase, suggesting futile cycle of incorporation and excision of wrong nucleotides. We tested incorporation of dU or rU, finding some evidence for both, but DNA-dU incorporation accelerates TLD only when intracellular [dUTP] is increased by the dut mutation. In the dut ung mutant, with increased DNA-dU incorporation and no DNA-dU excision, replication is in fact rescued even without dT, but TLD still occurs, suggesting different mechanisms. Finally, we found that continuous DNA synthesis during thymine starvation makes chromosomal DNA increasingly single-stranded, and even the dut ung defect does not completely block this ss-gap accumulation. We propose that instability of single-strand gaps underlies the pathology of thymine starvation.

Contribution of reactive oxygen species to thymineless death in Escherichia coli.

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Use of genetically modified bacteria for drug delivery in humans: Revisiting the safety aspect

The use of live, genetically modified bacteria as delivery vehicles for biologics is of considerable interest scientifically and has attracted significant commercial investment. We have pioneered the use of the commensal gut bacterium Bacteroides ovatus for the oral delivery of therapeutics to the gastrointestinal tract. Here we report on our investigations of the biological safety of engineered B. ovatus bacteria that includes the use of thymineless death as a containment strategy and the potential for the spread of transgenes in vivo in the mammalian gastrointestinal tract. We demonstrate the ability of GM-strains of Bacteroides to survive thymine starvation and overcome it through the exchange of genetic material. We also provide evidence for horizontal gene transfer in the mammalian gastrointestinal tract resulting in transgene-carrying wild type bacteria. These findings sound a strong note of caution on the employment of live genetically modified bacteria for the delivery of biologics.

Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs.

The methylfolate trap, a metabolic blockage associated with anemia, neural tube defects, Alzheimer’s dementia, cardiovascular diseases, and cancer, was discovered in the 1960s, linking the metabolism of folate, vitamin B12, methionine and homocysteine. However, the existence or physiological significance of this phenomenon has been unknown in bacteria, which synthesize folate de novo. Here we identify the methylfolate trap as a novel determinant of the bacterial intrinsic death by sulfonamides, antibiotics that block de novo folate synthesis. Genetic mutagenesis, chemical complementation, and metabolomic profiling revealed trap-mediated metabolic imbalances, which induced thymineless death, a phenomenon in which rapidly growing cells succumb to thymine starvation. Restriction of B12 bioavailability, required for preventing trap formation, using an “antivitamin B12” molecule, sensitized intracellular bacteria to sulfonamides. Since boosting the bactericidal activity of sulfonamides through methylfolate trap induction can be achieved in Gram-negative bacteria and mycobacteria, it represents a novel strategy to render these pathogens more susceptible to existing sulfonamides.

Fork restart protein, PriA, binds around oriC after depletion of nucleotide precursors: Replication fork arrest near the replication origin

Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at ∼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.

Thymineless Death Lives On: New Insights into a Classic Phenomenon.

The primary mechanisms by which bacteria lose viability when deprived of thymine have been elusive for over half a century. Early research focused on stalled replication forks and the deleterious effects of uracil incorporation into DNA from thymidine-deficient nucleotide pools. The initiation of the replication cycle and origin-proximal DNA degradation during thymine starvation have now been quantified via whole-genome microarrays and other approaches. These advances have fostered innovative models and informative experiments in bacteria since this topic was last reviewed. Given that thymineless death is similar in mammalian cells and that certain antibacterial and chemotherapeutic drugs elicit thymine deficiency, a mechanistic understanding of this phenomenon might have valuable biomedical applications.

Thymineless death, at the origin.

Thymineless death (TLD) in bacteria has been a focus of research for decades. Nevertheless, the advances in the last 5 years, with Escherichia coli as the model organism, have been outstanding. Independent research groups have presented compelling results that establish that the initiation of chromosome replication under thymine starvation is a key element in the scenario of TLD. Here we review the experimental results linking the initiation of replication to the lethality under thymine starvation and the proposed mechanisms by which TLD occurs. The concept of this relationship was ‘in the air,’ but approaches were not sufficiently developed to demonstrate the crucial role of DNA initiation in TLD. Genome-wide marker frequency analysis and Two Dimensional agarose gel electrophoresis have been critical methods employed to reveal that initiation events and the degradation of the oriC region occur during thymine starvation. The relationships between these events and TLD have established them to be the main underlying causes of the lethality under thymine starvation. Furthermore, we summarize additional important findings from the study of different mutant strains, which support the idea that the initiation of chromosomal replication and TLD are connected.

Perturbed states of the bacterial chromosome: a thymineless death case study.

Spatial patterns of transcriptional activity in the living genome of Escherichia coli represent one of the more peculiar aspects of the E. coli chromosome biology. Spatial transcriptional correlations can be observed throughout the chromosome, and their formation depends on the state of replication in the cell. The condition of thymine starvation leading to thymineless death (TLD) is at the “cross-roads” of replication and transcription. According to a current view, e.g., (Cagliero et al., 2014), one of the cellular objectives is to segregate the processes of transcription and replication in time and space. An ultimate segregation would take place when one process is inhibited and another is not, as it happens during thymine starvation, which results in numerous molecular and physiological abnormalities associated with TLD. One of such abnormalities is the loss of spatial correlations in the vicinity of the origin of replication. We review the transcriptional consequences of replication inhibition by thymine starvation in a context of the state of DNA template in the starved cells and opine about a possible significance of normal physiological coupling between the processes of replication and transcription.

Rifampicin suppresses thymineless death by blocking the transcription-dependent step of chromosome initiation.

Thymineless death (TLD), a phenomenon in which thymine auxotrophy becomes lethal when cells are starved of thymine, can be prevented by the presence of rifampicin, an RNA polymerase inhibitor. Several lines of evidence link TLD to chromosome initiation events. This suggests that rifampicin-mediated TLD suppression could be due to the inhibition of RNA synthesis required for DNA chromosomal initiation at oriC, although other mechanisms cannot be discarded. In this work, we show that the addition of different rifampicin concentrations to thymine-starved cells modulates TLD and chromosomal initiation capacity (ChIC). Time-lapse experiments find increasing levels of ChIC during thymine starvation correlated with the accumulation of simple-Y, double-Y and bubble arc replication intermediates at the oriC region as visualized by two-dimensional DNA agarose gel electrophoresis. None of these structures were observed following rifampicin addition or under genetic-physiological conditions that suppress TLD, indicating that abortive chromosome replication initiations under thymine starvation are crucial for this lethality. Significantly, the introduction of mioC and gid mutations which alter transcription levels around oriC, reduces ChIC and alleviates TLD. These results show that the impairment of transcription-dependent initiation caused by rifampicin addition, is responsible for TLD suppression. Our findings here may provide new avenues for the development of improved antibacterial treatments and chemotherapies based on thymine starvation-induced cell death.

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.

The association of DNA damage response and nucleotide level modulation with the antibacterial mechanism of the anti-folate drug Trimethoprim

BackgroundTrimethoprim is a widely prescribed antibiotic for a variety of bacterial infections. It belongs to a class of anti-metabolites - antifolates - which includes drugs used against malarial parasites and in cancer therapy. However, spread of bacterial resistance to the drug has severely hampered its clinical use and has necessitated further investigations into its mechanism of action and treatment regimen. Trimethoprim selectively starves bacterial cells for tetrahydrofolate, a vital cofactor necessary for the synthesis of several metabolites. The outcome (bacteriostatic or bactericidal) of such starvation, however, depends on the availability of folate-dependent metabolites in the growth medium. To characterize this dependency, we investigated in detail the regulatory and structural components of Escherichia coli cellular response to trimethoprim in controlled growth and supplementation conditions.ResultsWe surveyed transcriptional responses to trimethoprim treatment during bacteriostatic and bactericidal conditions and analyzed associated gene sets/pathways. Concurrent starvation of all folate dependent metabolites caused growth arrest, and this was accompanied by induction of general stress and stringent responses. Three gene sets were significantly associated with the bactericidal effect of TMP in different media including LB: genes of the SOS regulon, genes of the pyrimidine nucleotide biosynthetic pathway and members of the multiple antibiotic resistance (mar) regulon controlled by the MarR repressor. However, the SOS response was identified as the only universal transcriptional signature associated with the loss of viability by direct thymine starvation or by folate stress. We also used genome-wide gene knock-out screen to uncover means of sensitization of bacteria to the drug. We observed that among a number of candidate genes and pathways, the effect of knock-outs in the deoxyribose nucleotide salvage pathway, encoded by the deoCABD operon and under the control of the DeoR repressor, was most informative.ConclusionTranscriptional induction of DNA damage response is an essential feature of the bactericidal effect of trimethoprim. Either the observation of the transcriptional response or DNA damage itself, or both, is made possible by thymine starvation when other folate-dependent metabolites are not limited. The effect of DNA damage by the drug takes place prior to its bactericidal effect, at the beginning of the lag stage of the treatment. Mutations in the deoxyribose nucleotide salvage pathway can affect duration of the lag as well as the rate of killing. This information can be used to postulate certain mechanistic differences between direct thymine starvation in thymidylate synthase deficient mutants and thymine starvation by anti-folate inhibitors.

The dgt gene of Escherichia coli facilitates thymine utilization in thymine‐requiring strains

The Escherichia coli dGTP triphosphohydrolase (dGTPase) encoded by the dgt gene catalyses the hydrolysis of dGTP to deoxyguanosine and triphosphate. The recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. Here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the DeoD phosphorylase to deoxyribose-1-phosphate, the critical intermediate that enables uptake and utilization of thymine. Indeed, we observed that the minimal amount of thymine required for growth of thymine-requiring (thyA) strains decreased with increased expression level of the dgt gene. As expected, this dgt-mediated effect was dependent on the DeoD purine nucleoside phosphorylase. We also observed that thyA strains experience growth difficulties upon nutritional shift-up and that the dgt gene facilitates adaptation to the new growth conditions. Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate generation greatly exacerbated the severity of thymine starvation in enriched media, and under these conditions the dgt pathway becomes crucial in protecting the cells against thymineless death. Overall, our results suggest that the dgt-dependent pathway for deoxyribose-1-phosphate generation may operate under various cell conditions to provide deoxyribosyl donors.

Cytosine Methylation During Thymine Starvation: Effect of Nicotinamide

Cytosine Methylation During Thymine Starvation: Effect of Nicotinamide

DNA replication initiation as a key element in thymineless death

Thymine deprivation results in the loss of viability in cells from bacteria to eukaryotes. Numerous studies have identified a variety of molecular processes and cellular responses associated with thymineless death (TLD). It has been observed that TLD occurs in actively growing cells, and DNA damage and DNA recombination structures have been associated with cells undergoing TLD. We measured the loss of viability in thymine-starved cells differing in the number of overlapping replication cycles ( n), and we found that the magnitude of TLD correlates with the number of replication forks. By using pulsed field gel electrophoresis (PFGE), we determined the proportion of linear DNA (DSBs) and the amount of DNA remaining in the well after treatment with XbaI (nmDNA) under thymine starvation in the absence or presence of both rifampicin (suppressing TLD) and hydroxyurea (maintaining TLD). Our results indicate that DSBs and nmDNA are induced by thymine starvation, but they do not correlate with the lethality observed in the presence of the drugs. We asked whether TLD was related to chromosomal DNA initiation. DNA labeling experiments and flow cytometric analyses showed that new initiation events were induced under thymine starvation. These new DNA replication initiation events were inhibited in the presence of rifampicin but not in the presence of hydroxyurea, indicating that TLD correlates with the induction of new initiation events in Escherichia coli. In support of this finding, cells carrying a deletion of the datA site, in which DNA initiation is allowed in the presence of rifampicin, underwent TLD in the presence of rifampicin. We propose that thymineless-induced DNA initiation generates a fraction of DNA damage and/or nmDNA at origins that is critical for TLD. Our model provides new elements to be considered when testing mammalian chemotherapies that are based on the inhibition of thymidylate synthetase.

Thymineless death is associated with loss of essential genetic information from the replication origin

Thymine starvation results in a terminal cellular condition known as thymineless death (TLD), which is the basis of action for several common antibiotics and anticancer drugs. We characterized the onset and progression of TLD in Escherichia coli and found that DNA damage is the only salient property that distinguishes cells irreversibly senesced under thymine starvation from cells reversibly arrested by the nucleotide limitation. The damage is manifested as the relative loss of genetic material spreading outward from the replication origin: the extent of TLD correlates with the progression of damage. The reduced lethality in mutants deficient in the RecFOR/JQ repair pathway also correlates with the extent of damage, which explains most of the observed variance in cell killing. We propose that such spatially localized and persistent DNA damage is the consequence of transcription-dependent initiation of replication in the thymine-starved cells and may be the underlying cause of TLD.

The effect of 5-bromouracil treatment on diploid yeast cells.

The effect of treating diploid yeast cells of the constitution ade2/ + with 5-bromouracil has been studied. A synthetic medium containing aminopterin was used to create a condition of thymine starvation. 5-bromouracil or thymine were added to the blocked system to overcome the inhibition. It was expected, firstly, that incorporation of 5-BU or Th into the cells would result in growth and secondly, the effect of such an incorporation on mitotic crossing-over and/or gene conversion would result in an increase in sectored colonies. In the first series of experiments, the final concentration of Apt, 5-BU or Th was 100 μg/ml. In the second series two different concentrations of 5-BU were tested, viz. 50 μg/ml and 100 μg/ml. The treated suspensions were aerated for 40 hours at 28°C. The results demonstrate Apt-caused inhibition of DNA synthesis as anticipated. No significant increase in growth was observed between suspensions with Apt alone and with Apt and 5-BU or Th added. This indicates that neither 5-BU nor Th were incorporated into the cells of Saccharomyces cerevisiae when Apt was present. It was also observed that Apt-treated cells plated on agar medium formed significantly higher frequencies of sectored colonies than cells from control suspensions.

Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli.

Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

Role of DNA Replication and Repair in Thymineless Death in Escherichia coli

Inhibition of DNA replication with hydroxyurea during thymine starvation of Escherichia coli shows that active DNA synthesis is not required for thymineless death (TLD). Hydroxyurea experiments and thymine starvation of lexA3 and uvrA DNA repair mutants rule out unbalanced growth, the SOS response, and nucleotide excision repair as explanations for TLD.