Project description:dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving an E. coligpt optA1 strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included the purEK operon as well as the acrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions.IMPORTANCE Concentrations of the four precursors for DNA synthesis (2'-deoxynucleoside-5'-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition, E. coli cells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels.

Project description:UnlabelledOur laboratory recently discovered that Escherichia coli cells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for thymineless death (TLD). Conditions for specific dGTP starvation can be achieved by depriving an E. coli optA1 gpt strain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses of actively growing optA1 gpt cells subjected to hypoxanthine deprivation for increasing periods. The data show that upon Hx withdrawal, the optA1 gpt strain displays a diminished ability to derepress the de novo purine biosynthesis genes, likely due to internal guanine accumulation. The impairment in fully inducing the purR regulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS response was observed, supporting the concept of replication stress as a final cause of death. No evidence was observed in the starved cells for the participation of other stress responses, including the rpoS-mediated global stress response, reinforcing the lack of feedback of replication stress to the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient was observed for expression of genes located near the replication origin relative to those located toward the replication terminus.ImportanceControl of the supply of the building blocks (deoxynucleoside triphosphates [dNTPs]) for DNA replication is important for ensuring genome integrity and cell viability. When cells are starved specifically for one of the four dNTPs, dGTP, the process of DNA replication is disturbed in a manner that can lead to eventual death. In the present study, we investigated the transcriptional changes in the bacterium E. coli during dGTP starvation. The results show increasing DNA replication stress with an increased time of starvation, as evidenced by induction of the bacterial SOS system, as well as a notable lack of induction of other stress responses that could have saved the cells from cell death by slowing down cell growth.

Project description:BACKGROUND: Growth substrates, aerobic/anaerobic conditions, specific growth rate (?) etc. strongly influence Escherichia coli cell physiology in terms of cell size, biomass composition, gene and protein expression. To understand the regulation behind these different phenotype properties, it is useful to know carbon flux patterns in the metabolic network which are generally calculated by metabolic flux analysis (MFA). However, rarely is biomass composition determined and carbon balance carefully measured in the same experiments which could possibly lead to distorted MFA results and questionable conclusions. Therefore, we carried out both detailed carbon balance and biomass composition analysis in the same experiments for more accurate quantitative analysis of metabolism and MFA. RESULTS: We applied advanced continuous cultivation methods (A-stat and D-stat) to continuously monitor E. coli K-12 MG1655 flux and energy metabolism dynamic responses to change of ? and glucose-acetate co-utilisation. Surprisingly, a 36% reduction of ATP spilling was detected with increasing ? and carbon wasting to non-CO2 by-products under constant biomass yield. The apparent discrepancy between constant biomass yield and decline of ATP spilling could be explained by the rise of carbon wasting from 3 to 11% in the carbon balance which was revealed by the discovered novel excretion profile of E. coli pyrimidine pathway intermediates carbamoyl-phosphate, dihydroorotate and orotate. We found that carbon wasting patterns are dependent not only on ?, but also on glucose-acetate co-utilisation capability. Accumulation of these compounds was coupled to the two-phase acetate accumulation profile. Acetate overflow was observed in parallel with the reduction of TCA cycle and glycolysis fluxes, and induction of pentose phosphate pathway. CONCLUSIONS: It can be concluded that acetate metabolism is one of the major regulating factors of central carbon metabolism. More importantly, our model calculations with actual biomass composition and detailed carbon balance analysis in steady state conditions with -omics data comparison demonstrate the importance of a comprehensive systems biology approach for more advanced understanding of metabolism and carbon re-routing mechanisms potentially leading to more successful metabolic engineering.

Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

Project description:BackgroundMolecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.ResultsBy constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.ConclusionsBy eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.

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Slower growth of Escherichia coli leads to longer survival in carbon starvation due to a decrease in the maintenance rate

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