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Jack T. Pronk - One of the best experts on this subject based on the ideXlab platform.
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Chemostat-Based Micro-Array Analysis in Baker's Yeast
Advances in microbial physiology, 2009Co-Authors: Pascale Daran-lapujade, Jean-marc Daran, Antonius J. A. Van Maris, Johannes H. De Winde, Jack T. PronkAbstract:Chemostat cultivation of micro-organisms offers unique opportunities for experimental manipulation of individual environmental parameters at a fixed, controllable specific growth rate. Chemostat cultivation was originally developed as a tool to study quantitative aspects of microbial growth and metabolism. Renewed interest in this cultivation method is stimulated by the availability of high-information-density techniques for systemic analysis of microbial cultures, which require high reproducibility and careful experimental design. Genome-wide analysis of transcript levels with DNA micro-arrays is currently the most commonly applied of these high-information-density analysis tools for microbial gene expression. Based on published studies on the yeast Saccharomyces cerevisiae, a critical overview is presented of the possibilities and pitfalls associated with the combination of Chemostat cultivation and transcriptome analysis with DNA micro-arrays. After a brief introduction to Chemostat cultivation and micro-array analysis, key aspects of experimental design of Chemostat-based micro-array experiments are discussed. The main focus of this review is on key biological concepts that can be accessed by Chemostat-based micro-array analysis. These include effects of specific growth rate on transcriptional regulation, context-dependency of transcriptional responses, correlations between transcript profiles and contribution of the corresponding proteins to cellular function and fitness, and the analysis and application of evolutionary adaptation during prolonged Chemostat cultivation. It is concluded that, notwithstanding the incompatibility of Chemostat cultivation with high-throughput analysis, integration of Chemostat cultivation with micro-array analysis and other high-information-density analytical approaches (e.g. proteomics and metabolomics techniques) offers unique advantages in terms of reproducibility and experimental design in comparison with standard batch cultivation systems. Therefore, Chemostat cultivation and derived methods for controlled cultivation of micro-organisms are anticipated to become increasingly important in microbial physiology and systems biology.
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acclimation of saccharomyces cerevisiae to low temperature a Chemostat based transcriptome analysis
Molecular Biology of the Cell, 2007Co-Authors: Siew Leng Tai, Jack T. Pronk, Pascale Daranlapujade, Michael C Walsh, Jean-marc DaranAbstract:Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state Chemostats, grown at a fixed specific growth rate (0.03 h-1). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30°C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12°C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12°C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the Chemostat- based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.
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Chemostat cultivation as a tool for studies on sugar transport in yeasts
Microbiological sciences, 1994Co-Authors: Ruud A. Weusthuis, Jack T. Pronk, P. Van Den BroekAbstract:Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (q(s)) in sugar-limited Chemostat cultures. A straightforward way to manipulate q, is variation of the dilution rate, which, in substrate-limited Chemostat cultures, is equal to the specific growth rate. Alternatively, q(s) can be varied independently of the growth rate by mixed substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited Chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under these conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited Chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in Chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.
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Chemostat cultivation as a tool for studies on sugar transport in yeasts.
Microbiological reviews, 1994Co-Authors: Ruud A. Weusthuis, Jack T. Pronk, P. Van Den Broek, J.p. Van DijkenAbstract:Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited Chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited Chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited Chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited Chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in Chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.
Jean-marc Daran - One of the best experts on this subject based on the ideXlab platform.
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Degeneration of penicillin production in ethanol-limited Chemostat cultivations of Penicillium chrysogenum: A systems biology approach
BMC Systems Biology, 2011Co-Authors: Rutger D Douma, Joana M. Batista, Kai M. Touw, Paul Klaassen, Roel A. L. Bovenberg, Arjen M. Krikken, Jan A.k.w. Kiel, Tania Veiga, Zheng Zhao, Jean-marc DaranAbstract:Background In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example Chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited Chemostat cultivations. Results During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited Chemostats did only result in a slight recovery of penicillin production. Conclusions Our findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.
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Chemostat-Based Micro-Array Analysis in Baker's Yeast
Advances in microbial physiology, 2009Co-Authors: Pascale Daran-lapujade, Jean-marc Daran, Antonius J. A. Van Maris, Johannes H. De Winde, Jack T. PronkAbstract:Chemostat cultivation of micro-organisms offers unique opportunities for experimental manipulation of individual environmental parameters at a fixed, controllable specific growth rate. Chemostat cultivation was originally developed as a tool to study quantitative aspects of microbial growth and metabolism. Renewed interest in this cultivation method is stimulated by the availability of high-information-density techniques for systemic analysis of microbial cultures, which require high reproducibility and careful experimental design. Genome-wide analysis of transcript levels with DNA micro-arrays is currently the most commonly applied of these high-information-density analysis tools for microbial gene expression. Based on published studies on the yeast Saccharomyces cerevisiae, a critical overview is presented of the possibilities and pitfalls associated with the combination of Chemostat cultivation and transcriptome analysis with DNA micro-arrays. After a brief introduction to Chemostat cultivation and micro-array analysis, key aspects of experimental design of Chemostat-based micro-array experiments are discussed. The main focus of this review is on key biological concepts that can be accessed by Chemostat-based micro-array analysis. These include effects of specific growth rate on transcriptional regulation, context-dependency of transcriptional responses, correlations between transcript profiles and contribution of the corresponding proteins to cellular function and fitness, and the analysis and application of evolutionary adaptation during prolonged Chemostat cultivation. It is concluded that, notwithstanding the incompatibility of Chemostat cultivation with high-throughput analysis, integration of Chemostat cultivation with micro-array analysis and other high-information-density analytical approaches (e.g. proteomics and metabolomics techniques) offers unique advantages in terms of reproducibility and experimental design in comparison with standard batch cultivation systems. Therefore, Chemostat cultivation and derived methods for controlled cultivation of micro-organisms are anticipated to become increasingly important in microbial physiology and systems biology.
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acclimation of saccharomyces cerevisiae to low temperature a Chemostat based transcriptome analysis
Molecular Biology of the Cell, 2007Co-Authors: Siew Leng Tai, Jack T. Pronk, Pascale Daranlapujade, Michael C Walsh, Jean-marc DaranAbstract:Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state Chemostats, grown at a fixed specific growth rate (0.03 h-1). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30°C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12°C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12°C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the Chemostat- based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.
Frederic Mazenc - One of the best experts on this subject based on the ideXlab platform.
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Stability analysis of mathematical model of competition in a chain of Chemostats in series with delay
Applied Mathematical Modelling, 2019Co-Authors: Frederic Mazenc, Silviu-iulian Niculescu, Gonzalo RobledoAbstract:We study a nonlinear system of differential delay equations describing a model of a chain of two Chemostats, where one contains two microbial species in competition for a single limiting nutrient and receives an external input of the less advantaged competitor, which is cultivated in an external Chemostat. We obtain sufficient conditions ensuring coexistence of all the species in competition which consist in upper delay bounds. © 2019 Elsevier Inc.
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Stability and stabilization for models of Chemostats with multiple limiting substrates
Journal of Biological Dynamics, 2012Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study Chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations.
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stabilization of a Chemostat model with haldane growth functions and a delay in the measurements
Automatica, 2010Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:The stabilization of equilibria in Chemostats with measurement delays is a complex and challenging problem, and is of significant ongoing interest in bioengineering and population dynamics. In this paper, we solve an output feedback stabilization problem for Chemostat models having two species, one limiting substrate, and either Haldane or Monod growth functions. Our feedback stabilizers depend on a given linear combination of the species concentrations, which are both measured with a constant time delay. The values of the delays are unknown. Instead, one only knows an upper bound on the delays, and we allow the upper bound to be arbitrarily large. The stabilizing feedback depends on the known upper bound for the delays as well. Our work is based on a Lyapunov-Krasovskii argument.
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Further results on stabilization of periodic trajectories for a Chemostat with two species
IEEE Transactions on Automatic Control, 2008Co-Authors: Frederic Mazenc, Michael Malisoff, Jérôme HarmandAbstract:We discuss an important class of problems involving the tracking of prescribed trajectories in the Chemostat model. We provide new tracking results for Chemostats with two species and one limiting substrate, based on Lyapunov function methods. In particular, we use a linear feedback control of the dilution rate and an appropriate time-varying substrate input concentration to produce a locally exponentially stable oscillatory behavior. This means that all trajectories for the nutrient and corresponding species concentrations in the closed loop Chemostat that stay near the oscillatory reference trajectory are attracted to the reference trajectory exponentially fast. We also obtain a globally stable oscillatory reference trajectory for the species concentrations, using a nonlinear feedback control depending on the dilution rate and the substrate input concentration. This guarantees that all trajectories for the closed loop Chemostat dynamics are attracted to the reference trajectory. Finally, we construct an explicit Lyapunov function for the corresponding global error dynamics. We demonstrate the efficacy of our method in a simulation.
Michael Malisoff - One of the best experts on this subject based on the ideXlab platform.
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Stability and stabilization for models of Chemostats with multiple limiting substrates
Journal of Biological Dynamics, 2012Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:We study Chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled Chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for Chemostats with one limiting nutrient. We demonstrate our methods in simulations.
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stabilization of a Chemostat model with haldane growth functions and a delay in the measurements
Automatica, 2010Co-Authors: Frederic Mazenc, Michael MalisoffAbstract:The stabilization of equilibria in Chemostats with measurement delays is a complex and challenging problem, and is of significant ongoing interest in bioengineering and population dynamics. In this paper, we solve an output feedback stabilization problem for Chemostat models having two species, one limiting substrate, and either Haldane or Monod growth functions. Our feedback stabilizers depend on a given linear combination of the species concentrations, which are both measured with a constant time delay. The values of the delays are unknown. Instead, one only knows an upper bound on the delays, and we allow the upper bound to be arbitrarily large. The stabilizing feedback depends on the known upper bound for the delays as well. Our work is based on a Lyapunov-Krasovskii argument.
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Further results on stabilization of periodic trajectories for a Chemostat with two species
IEEE Transactions on Automatic Control, 2008Co-Authors: Frederic Mazenc, Michael Malisoff, Jérôme HarmandAbstract:We discuss an important class of problems involving the tracking of prescribed trajectories in the Chemostat model. We provide new tracking results for Chemostats with two species and one limiting substrate, based on Lyapunov function methods. In particular, we use a linear feedback control of the dilution rate and an appropriate time-varying substrate input concentration to produce a locally exponentially stable oscillatory behavior. This means that all trajectories for the nutrient and corresponding species concentrations in the closed loop Chemostat that stay near the oscillatory reference trajectory are attracted to the reference trajectory exponentially fast. We also obtain a globally stable oscillatory reference trajectory for the species concentrations, using a nonlinear feedback control depending on the dilution rate and the substrate input concentration. This guarantees that all trajectories for the closed loop Chemostat dynamics are attracted to the reference trajectory. Finally, we construct an explicit Lyapunov function for the corresponding global error dynamics. We demonstrate the efficacy of our method in a simulation.
David Gresham - One of the best experts on this subject based on the ideXlab platform.
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Single-cell copy number variant detection reveals the dynamics and diversity of adaptation.
PLOS Biology, 2018Co-Authors: Stephanie Lauer, Nathan Brandt, Grace Avecilla, Pieter Spealman, Gunjan Sethia, Sasha F. Levy, David GreshamAbstract:Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in Chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited Chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1’s proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited Chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 102–104 independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18–21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.
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The functional basis of adaptive evolution in Chemostats.
FEMS microbiology reviews, 2014Co-Authors: David Gresham, Jungeui HongAbstract:Two of the central problems in biology are determining the molecular basis of adaptive evolution and understanding how cells regulate their growth. The Chemostat is a device for culturing cells that provides great utility in tackling both of these problems: it enables precise control of the selective pressure under which organisms evolve and it facilitates experimental control of cell growth rate. The aim of this review is to synthesize results from studies of the functional basis of adaptive evolution in long-term Chemostat selections using Escherichia coli and Saccharomyces cerevisiae . We describe the principle of the Chemostat, provide a summary of studies of experimental evolution in Chemostats, and use these studies to assess our current understanding of selection in the Chemostat. Functional studies of adaptive evolution in Chemostats provide a unique means of interrogating the genetic networks that control cell growth, which complements functional genomic approaches and quantitative trait loci (QTL) mapping in natural populations. An integrated approach to the study of adaptive evolution that accounts for both molecular function and evolutionary processes is critical to advancing our understanding of evolution. By renewing efforts to integrate these two research programs, experimental evolution in Chemostats is ideally suited to extending the functional synthesis to the study of genetic networks.
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The Use of Chemostats in Microbial Systems Biology
Journal of visualized experiments : JoVE, 2013Co-Authors: Naomi Ziv, Nathan Brandt, David GreshamAbstract:Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The Chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations Chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of Chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of Chemostats in biological research.