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

In vivo dual RNA-seq reveals that neutrophil recruitment underlies differential tissue tropism of Streptococcus pneumoniae.

Streptococcus pneumoniae is a genetically diverse human-adapted pathogen commonly carried asymptomatically in the nasopharynx. We have recently shown that a single nucleotide polymorphism (SNP) in the raffinose pathway regulatory gene rafR accounts for a difference in the capacity of clonally-related strains to cause localised versus systemic infection. Using dual RNA-seq, we show that this SNP affects expression of bacterial genes encoding multiple sugar transporters, and fine-tunes carbohydrate metabolism, along with extensive rewiring of host transcriptional responses to infection, particularly expression of genes encoding cytokine and chemokine ligands and receptors. The data predict a crucial role for differential neutrophil recruitment (confirmed by in vivo neutrophil depletion and IL-17 neutralization) indicating that early detection of bacteria by the host in the lung environment is crucial for effective clearance. Thus, dual RNA-seq provides a powerful tool for understanding complex host-pathogen interactions and reveals how a single bacterial SNP can drive differential disease outcomes

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus.

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc <sub>2</sub> DAG. By contrast, in cells lacking LtaA, the flippase of Glc <sub>2</sub> DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus

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

Individual differences in puberty onset in girls: Bayesian estimation of heritabilities and genetic correlations

We report heritabilities for individual differences in female pubertal development at the age of 12. Tanner data on breast and pubic hair development in girls and data on menarche were obtained from a total of 184 pairs of monozygotic and dizygotic twins. Genetic correlations were estimated to determine to what extent the same genes are involved in different aspects of physical development in puberty. A Bayesian estimation approach was taken, using Markovchain Monte Carlo simulation to estimate model parameters. All three phenotypes were to a significant extent heritable and showed high genetic correlations, suggesting that a common set of genes is involved in the timing of puberty in general. However, gonadarche (menarche and breast development) and adrenarche (pubic hair) are affected by different environmental factors, which does not support the three phenotypes to be regarded as indicators of a unitary physiological factor. © 2006 Springer Science+Business Media, Inc

Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites

Bet Hedging in Yeast by Heterogeneous, Age-Correlated Expression of a Stress Protectant

A new experimental approach reveals a bet-hedging strategy in unstressed, clonal yeast cells, whereby they adopt a range of growth states that correlate with expression of a trehalose-synthesis regulator and predict resistance to future stress

Adaptable Functionality of Transcriptional Feedback in Bacterial Two-Component Systems

A widespread mechanism of bacterial signaling occurs through two-component systems, comprised of a sensor histidine kinase (SHK) and a transcriptional response regulator (RR). The SHK activates RR by phosphorylation. The most common two-component system structure involves expression from a single operon, the transcription of which is activated by its own phosphorylated RR. The role of this feedback is poorly understood, but it has been associated with an overshooting kinetic response and with fast recovery of previous interrupted signaling events in different systems. Mathematical models show that overshoot is only attainable with negative feedback that also improves response time. Our models also predict that fast recovery of previous interrupted signaling depends on high accumulation of SHK and RR, which is more likely in a positive feedback regime. We use Monte Carlo sampling of the parameter space to explore the range of attainable model behaviors. The model predicts that the effective feedback sign can change from negative to positive depending on the signal level. Variations in two-component system architectures and parameters may therefore have evolved to optimize responses in different bacterial lifestyles. We propose a conceptual model where low signal conditions result in a responsive system with effectively negative feedback while high signal conditions with positive feedback favor persistence of system output

A Microscope Automated Fluidic System to Study Bacterial Processes in Real Time

Most time lapse microscopy experiments studying bacterial processes ie growth, progression through the cell cycle and motility have been performed on thin nutrient agar pads. An important limitation of this approach is that dynamic perturbations of the experimental conditions cannot be easily performed. In eukaryotic cell biology, fluidic approaches have been largely used to study the impact of rapid environmental perturbations on live cells and in real time. However, all these approaches are not easily applicable to bacterial cells because the substrata are in all cases specific and also because microfluidics nanotechnology requires a complex lithography for the study of micrometer sized bacterial cells. In fact, in many cases agar is the experimental solid substratum on which bacteria can move or even grow. For these reasons, we designed a novel hybrid micro fluidic device that combines a thin agar pad and a custom flow chamber. By studying several examples, we show that this system allows real time analysis of a broad array of biological processes such as growth, development and motility. Thus, the flow chamber system will be an essential tool to study any process that take place on an agar surface at the single cell level

Mapping the Environmental Fitness Landscape of a Synthetic Gene Circuit

Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Traditional biological measurement techniques have revealed the connection between the environment and fitness based on the gene expression mean. Yet, recently it became clear that cells with identical genomes exposed to the same environment can differ dramatically from the population average in their gene expression and division rate (individual fitness). For cell populations with bimodal gene expression, this difference is particularly pronounced, and may involve stochastic transitions between two cellular states that form distinct sub-populations. Currently it remains unclear how a cell population's growth rate and its subpopulation fractions emerge from the molecular-level kinetics of gene networks and the division rates of single cells. To address this question we developed and quantitatively characterized an inducible, bistable synthetic gene circuit controlling the expression of a bifunctional antibiotic resistance gene in Saccharomyces cerevisiae. Following fitness and fluorescence measurements in two distinct environments (inducer alone and antibiotic alone), we applied a computational approach to predict cell population fitness and subpopulation fractions in the combination of these environments based on stochastic cellular movement in gene expression space and fitness space. We found that knowing the fitness and nongenetic (cellular) memory associated with specific gene expression states were necessary for predicting the overall fitness of cell populations in combined environments. We validated these predictions experimentally and identified environmental conditions that defined a “sweet spot” of drug resistance. These findings may provide a roadmap for connecting the molecular-level kinetics of gene networks to cell population fitness in well-defined environments, and may have important implications for phenotypic variability of drug resistance in natural settings