What makes the lac-pathway switch: identifying the fluctuations that trigger phenotype switching in gene regulatory systems

Multistable gene regulatory systems sustain different levels of gene expression under identical external conditions. Such multistability is used to encode phenotypic states in processes including nutrient uptake and persistence in bacteria, fate selection in viral infection, cell cycle control, and development. Stochastic switching between different phenotypes can occur as the result of random fluctuations in molecular copy numbers of mRNA and proteins arising in transcription, translation, transport, and binding. However, which component of a pathway triggers such a transition is generally not known. By linking single-cell experiments on the lactose-uptake pathway in E. coli to molecular simulations, we devise a general method to pinpoint the particular fluctuation driving phenotype switching and apply this method to the transition between the uninduced and induced states of the lac genes. We find that the transition to the induced state is not caused only by the single event of lac-repressor unbinding, but depends crucially on the time period over which the repressor remains unbound from the lac-operon. We confirm this notion in strains with a high expression level of the repressor (leading to shorter periods over which the lac-operon remains unbound), which show a reduced switching rate. Our techniques apply to multi-stable gene regulatory systems in general and allow to identify the molecular mechanisms behind stochastic transitions in gene regulatory circuits.Comment: Version

Pulsed Feedback Defers Cellular Differentiation

Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable “polyphasic” positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a “timer” that operates over timescales much longer than a cell cycle

Ultrasensitivity of the Bacillus subtilis sporulation decision

Starving Bacillus subtilis cells execute a gene expression program resulting in the formation of stress-resistant spores. Sporulation master regulator, Spo0A, is activated by a phosphorelay and controls the expression of a multitude of genes, including the forespore- specific sigma factor σF and the mother cell-specific sigma factor σE. Identification of the system-level mechanism of the sporulation decision is hindered by a lack of direct control over Spo0A activity. This limitation can be overcome by using a synthetic system in which Spo0A activation is controlled by inducing expression of phosphorelay kinase KinA. This induction results in a switch-like increase in the number of sporulating cells at a threshold of KinA. Using a combination of mathematical modeling and single-cell microscopy, we investigate the origin and physiological significance of this ultrasensitive threshold. The results indicate that the phosphorelay is unable to achieve a sufficiently fast and ultrasensitive response via its positive feedback architecture, suggesting that the sporulation decision is made downstream. In contrast, activation of σF in the forespore and of σE in the mother cell compartments occurs via a cascade of coherent feed-forward loops, and thereby can produce fast and ultrasensitive responses as a result of KinA induction. Unlike σF activation, σE activation in the mother cell compartment only occurs above the KinA threshold, resulting in completion of sporulation. Thus, ultrasensitive σE activation explains the KinA threshold for sporulation induction. We therefore infer that under uncertain conditions, cells initiate sporulation but postpone making the sporulation decision to average stochastic fluctuations and to achieve a robust population response

Acute Activation of Metabolic Syndrome Components in Pediatric Acute Lymphoblastic Leukemia Patients Treated with Dexamethasone

Although dexamethasone is highly effective in the treatment of pediatric acute lymphoblastic leukemia (ALL), it can cause serious metabolic side effects. Because studies regarding the effects of dexamethasone are limited by their small scale, we prospectively studied the direct effects of treating pediatric ALL with dexamethasone administration with respect to activation of components of metabolic syndrome (MetS); in addition, we investigated whether these side effects were correlated with the level of dexamethasone. Fifty pediatric patients (3-16 years of age) with ALL were studied during a 5-day dexamethasone course during the maintenance phase of the Dutch Childhood Oncology Group ALL-10 and ALL-11 protocols. Fasting insulin, glucose, total cholesterol, HDL, LDL, and triglycerides levels were measured at baseline (before the start of dexamethasone; T1) and on the fifth day of treatment (T2). Dexamethasone trough levels were measured at T2. We found that dexamethasone treatment significantly increased the following fasting serum levels (P3.4) from 8% to 85%(P</p

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae.

Genome-wide screens have discovered a large set of essential genes in the opportunistic human pathogen &lt;i&gt;Streptococcus pneumoniae&lt;/i&gt; However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn-seq), we refined the list of essential genes in &lt;i&gt;S. pneumoniae&lt;/i&gt; serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high-content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi-based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp-proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP-dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets

Модель взаємовідносин між державою та фінансово-промисловими групами для різних бізнес-систем

Мета дослідження - визначення пріоритетів та стратегічних напрямків формування ефективної моделі взаємодії української держави з вітчизняними фінансово-промисловими групами в сучасних умовах економічного розвитку

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Acute activation of metabolic syndrome components in pediatric acute lymphoblastic leukemia patients treated with dexamethasone

Although dexamethasone is highly effective in the treatment of pediatric acute lymphoblastic leukemia (ALL), it can cause serious metabolic side effects. Because studies regarding the effects of dexamethasone are limited by their small scale, we prospectively studied the direct effects of treating pediatric ALL with dexamethasone administration with respect to activation of components of metabolic syndrome (MetS); in addition, we investigated whether these side effects were correlated with the level of dexamethasone. Fifty pediatric patients (3-16 years of age) with ALL were studied during a 5-day dexamethasone course during the maintenance phase of the Dutch Childhood Oncology Group ALL-10 and ALL-11 protocols. Fasting insulin, glucose, total cholesterol, HDL, LDL, and triglycerides levels were measured at baseline (before the start of dexamethasone; T1) and on the fifth day of treatment (T2). Dexamethasone trough levels were measured at T2. We found that dexamethasone treatment significantly increased the following fasting serum levels (P3.4) from 8% to 85% (P<0.01). Dexamethasone treatment also significantly increased the diastolic and systolic blood pressure. Lastly, dexamethasone trough levels (N = 24) were directly correlated with high glucose levels at T2, but not with other parameters. These results indicate that dexamethasone treatment acutely induces three components of the MetS. Together with the weight gain typically associated with dexamethasone treatment, these factors may contribute to the higher prevalence of MetS and cardiovascular risk among survivors of childhood leukemia who received dexamethasone treatment

Spo0A∼P Imposes a Temporal Gate for the Bimodal Expression of Competence in Bacillus subtilis

ComK transcriptionally controls competence for the uptake of transforming DNA in Bacillus subtilis. Only 10%–20% of the cells in a clonal population are randomly selected for competence. Because ComK activates its own promoter, cells exceeding a threshold amount of ComK trigger a positive feedback loop, transitioning to the competence ON state. The transition rate increases to a maximum during the approach to stationary phase and then decreases, with most cells remaining OFF. The average basal rate of comK transcription increases transiently, defining a window of opportunity for transitions and accounting for the heterogeneity of competent populations. We show that as the concentration of the response regulator Spo0A∼P increases during the entry to stationary phase it first induces comK promoter activity and then represses it by direct binding. Spo0A∼P activates by antagonizing the repressor, Rok. This amplifies an inherent increase in basal level comK promoter activity that takes place during the approach to stationary phase and is a general feature of core promoters, serving to couple the probability of competence transitions to growth rate. Competence transitions are thus regulated by growth rate and temporally controlled by the complex mechanisms that govern the formation of Spo0A∼P. On the level of individual cells, the fate-determining noise for competence is intrinsic to the comK promoter. This overall mechanism has been stochastically simulated and shown to be plausible. Thus, a deterministic mechanism modulates an inherently stochastic process

Clinical and self-reported markers of reproductive function in female survivors of childhood Hodgkin lymphoma

Purpose: To evaluate the impact of treatment for Hodgkin lymphoma (HL) on clinical reproductive markers and pregnancy outcomes.Methods: This study was embedded within the DCOG LATER-VEVO study; a Dutch, multicenter, retrospective cohort study between 2004 and 2014. Serum anti-Müllerian hormone (AMH), follicle stimulating hormone (FSH), inhibin B, antral follicle count (AFC), and self-reported (first) pregnancy outcomes were evaluated in female childhood HL survivors and controls.Results: 84 HL survivors and 798 controls were included, aged 29.6 and 32.7 years old at time of assessment. Median age at HL diagnosis was 13.4 years. Cyclophosphamide equivalent dose (CED-score) exceeded 6000 mg/m2 in 56 women and 14 survivors received pelvic irradiation.All clinical markers were significantly deteriorated in survivors (odds-ratio for low AMH (&lt; p10) 10.1 [95% CI 4.9; 20.6]; low AFC (&lt; p10) 4.6 [95% CI 2.1; 9.9]; elevated FSH (&gt; 10 IU/l) 15.3 [95% CI 5.7; 41.1], low Inhibin B (&lt; 20 ng/l) 3.6 [95% CI 1.7; 7.7], p &lt; 0.001). Pregnancy outcomes were comparable between survivors and controls (± 80% live birth, ± 20% miscarriage). However, survivors were significantly younger at first pregnancy (27.0 years vs 29.0 years, P = 0.04). Adjusted odds-ratio for time to pregnancy &gt; 12 months was 2.5 [95% CI 1.1; 5.6] in survivors, p = 0.031. Adverse outcomes were specifically present after treatment with procarbazine and higher CED-score.Conclusion: HL survivors appear to have an impaired ovarian reserve. However, chance to achieve pregnancy seems reassuring at a young age. Additional follow-up studies are needed to assess fertile life span and reproductive potential of HL survivors, in particular for current HL treatments that are hypothesized to be less gonadotoxic.</p