TY - JOUR AB - In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype. AU - Guet, Calin C AU - Bruneaux, L AU - Oikonomou, P AU - Aldana, M AU - Cluzel, P ID - 12478 JF - Frontiers in Microbiology TI - Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression VL - 14 ER - TY - JOUR AB - Understanding and characterising biochemical processes inside single cells requires experimental platforms that allow one to perturb and observe the dynamics of such processes as well as computational methods to build and parameterise models from the collected data. Recent progress with experimental platforms and optogenetics has made it possible to expose each cell in an experiment to an individualised input and automatically record cellular responses over days with fine time resolution. However, methods to infer parameters of stochastic kinetic models from single-cell longitudinal data have generally been developed under the assumption that experimental data is sparse and that responses of cells to at most a few different input perturbations can be observed. Here, we investigate and compare different approaches for calculating parameter likelihoods of single-cell longitudinal data based on approximations of the chemical master equation (CME) with a particular focus on coupling the linear noise approximation (LNA) or moment closure methods to a Kalman filter. We show that, as long as cells are measured sufficiently frequently, coupling the LNA to a Kalman filter allows one to accurately approximate likelihoods and to infer model parameters from data even in cases where the LNA provides poor approximations of the CME. Furthermore, the computational cost of filtering-based iterative likelihood evaluation scales advantageously in the number of measurement times and different input perturbations and is thus ideally suited for data obtained from modern experimental platforms. To demonstrate the practical usefulness of these results, we perform an experiment in which single cells, equipped with an optogenetic gene expression system, are exposed to various different light-input sequences and measured at several hundred time points and use parameter inference based on iterative likelihood evaluation to parameterise a stochastic model of the system. AU - Davidović, Anđela AU - Chait, Remy P AU - Batt, Gregory AU - Ruess, Jakob ID - 10939 IS - 3 JF - PLoS Computational Biology SN - 1553-734X TI - Parameter inference for stochastic biochemical models from perturbation experiments parallelised at the single cell level VL - 18 ER - TY - JOUR AB - Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin–antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine–cytosine–adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5′-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes. AU - Nikolic, Nela AU - Sauert, Martina AU - Albanese, Tanino G. AU - Moll, Isabella ID - 11713 JF - BMC Research Notes KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine SN - 1756-0500 TI - Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli VL - 15 ER - TY - JOUR AB - Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10–20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought. AU - Lagator, Mato AU - Sarikas, Srdjan AU - Steinrueck, Magdalena AU - Toledo-Aparicio, David AU - Bollback, Jonathan P AU - Guet, Calin C AU - Tkačik, Gašper ID - 10736 JF - eLife TI - Predicting bacterial promoter function and evolution from random sequences VL - 11 ER - TY - JOUR AB - Several promising strategies based on combining or cycling different antibiotics have been proposed to increase efficacy and counteract resistance evolution, but we still lack a deep understanding of the physiological responses and genetic mechanisms that underlie antibiotic interactions and the clinical applicability of these strategies. In antibiotic-exposed bacteria, the combined effects of physiological stress responses and emerging resistance mutations (occurring at different time scales) generate complex and often unpredictable dynamics. In this Review, we present our current understanding of bacterial cell physiology and genetics of responses to antibiotics. We emphasize recently discovered mechanisms of synergistic and antagonistic drug interactions, hysteresis in temporal interactions between antibiotics that arise from microbial physiology and interactions between antibiotics and resistance mutations that can cause collateral sensitivity or cross-resistance. We discuss possible connections between the different phenomena and indicate relevant research directions. A better and more unified understanding of drug and genetic interactions is likely to advance antibiotic therapy. AU - Römhild, Roderich AU - Bollenbach, Mark Tobias AU - Andersson, Dan I. ID - 10812 JF - Nature Reviews Microbiology KW - General Immunology and Microbiology KW - Microbiology KW - Infectious Diseases SN - 1740-1526 TI - The physiology and genetics of bacterial responses to antibiotic combinations VL - 20 ER - TY - JOUR AB - The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role. AU - Glover, Georgina AU - Voliotis, Margaritis AU - Łapińska, Urszula AU - Invergo, Brandon M. AU - Soanes, Darren AU - O’Neill, Paul AU - Moore, Karen AU - Nikolic, Nela AU - Petrov, Peter AU - Milner, David S. AU - Roy, Sumita AU - Heesom, Kate AU - Richards, Thomas A. AU - Tsaneva-Atanasova, Krasimira AU - Pagliara, Stefano ID - 11339 JF - Communications Biology TI - Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells VL - 5 ER - TY - JOUR AB - A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease. AU - Tomasek, Kathrin AU - Leithner, Alexander F AU - Glatzová, Ivana AU - Lukesch, Michael S. AU - Guet, Calin C AU - Sixt, Michael K ID - 11843 JF - eLife TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 VL - 11 ER - TY - JOUR AB - Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in Escherichia coli. We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales. AU - Tomanek, Isabella AU - Guet, Calin C ID - 12333 JF - eLife TI - Adaptation dynamics between copynumber and point mutations VL - 11 ER - TY - GEN AB - Copy-number and point mutations form the basis for most evolutionary novelty through the process of gene duplication and divergence. While a plethora of genomic sequence data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic system that allows us to distinguish copy-number and point mutations, we study their early and transient adaptive dynamics in real-time in Escherichia coli. We find two qualitatively different routes of adaptation depending on the level of functional improvement selected for: In conditions of high gene expression demand, the two types of mutations occur as a combination. Under low gene expression demand, negative epistasis between the two types of mutations renders them mutually exclusive. Thus, owing to their higher frequency, adaptation is dominated by copy-number mutations. Ultimately, due to high rates of reversal and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation but also constrain sequence divergence over evolutionary time scales. AU - Tomanek, Isabella AU - Guet, Calin C ID - 12339 TI - Flow cytometry YFP and CFP data and deep sequencing data of populations evolving in galactose ER - TY - JOUR AU - Römhild, Roderich AU - Andersson, Dan I. ID - 9046 IS - 1 JF - PLoS Pathogens SN - 15537366 TI - Mechanisms and therapeutic potential of collateral sensitivity to antibiotics VL - 17 ER - TY - JOUR AB - Gene expression is regulated by the set of transcription factors (TFs) that bind to the promoter. The ensuing regulating function is often represented as a combinational logic circuit, where output (gene expression) is determined by current input values (promoter bound TFs) only. However, the simultaneous arrival of TFs is a strong assumption, since transcription and translation of genes introduce intrinsic time delays and there is no global synchronisation among the arrival times of different molecular species at their targets. We present an experimentally implementable genetic circuit with two inputs and one output, which in the presence of small delays in input arrival, exhibits qualitatively distinct population-level phenotypes, over timescales that are longer than typical cell doubling times. From a dynamical systems point of view, these phenotypes represent long-lived transients: although they converge to the same value eventually, they do so after a very long time span. The key feature of this toy model genetic circuit is that, despite having only two inputs and one output, it is regulated by twenty-three distinct DNA-TF configurations, two of which are more stable than others (DNA looped states), one promoting and another blocking the expression of the output gene. Small delays in input arrival time result in a majority of cells in the population quickly reaching the stable state associated with the first input, while exiting of this stable state occurs at a slow timescale. In order to mechanistically model the behaviour of this genetic circuit, we used a rule-based modelling language, and implemented a grid-search to find parameter combinations giving rise to long-lived transients. Our analysis shows that in the absence of feedback, there exist path-dependent gene regulatory mechanisms based on the long timescale of transients. The behaviour of this toy model circuit suggests that gene regulatory networks can exploit event timing to create phenotypes, and it opens the possibility that they could use event timing to memorise events, without regulatory feedback. The model reveals the importance of (i) mechanistically modelling the transitions between the different DNA-TF states, and (ii) employing transient analysis thereof. AU - Petrov, Tatjana AU - Igler, Claudia AU - Sezgin, Ali AU - Henzinger, Thomas A AU - Guet, Calin C ID - 9647 JF - Theoretical Computer Science SN - 0304-3975 TI - Long lived transients in gene regulation VL - 893 ER - TY - JOUR AB - Attachment of adhesive molecules on cell culture surfaces to restrict cell adhesion to defined areas and shapes has been vital for the progress of in vitro research. In currently existing patterning methods, a combination of pattern properties such as stability, precision, specificity, high-throughput outcome, and spatiotemporal control is highly desirable but challenging to achieve. Here, we introduce a versatile and high-throughput covalent photoimmobilization technique, comprising a light-dose-dependent patterning step and a subsequent functionalization of the pattern via click chemistry. This two-step process is feasible on arbitrary surfaces and allows for generation of sustainable patterns and gradients. The method is validated in different biological systems by patterning adhesive ligands on cell-repellent surfaces, thereby constraining the growth and migration of cells to the designated areas. We then implement a sequential photopatterning approach by adding a second switchable patterning step, allowing for spatiotemporal control over two distinct surface patterns. As a proof of concept, we reconstruct the dynamics of the tip/stalk cell switch during angiogenesis. Our results show that the spatiotemporal control provided by our “sequential photopatterning” system is essential for mimicking dynamic biological processes and that our innovative approach has great potential for further applications in cell science. AU - Zisis, Themistoklis AU - Schwarz, Jan AU - Balles, Miriam AU - Kretschmer, Maibritt AU - Nemethova, Maria AU - Chait, Remy P AU - Hauschild, Robert AU - Lange, Janina AU - Guet, Calin C AU - Sixt, Michael K AU - Zahler, Stefan ID - 9822 IS - 30 JF - ACS Applied Materials and Interfaces SN - 19448244 TI - Sequential and switchable patterning for studying cellular processes under spatiotemporal control VL - 13 ER - TY - JOUR AB - Evolutionary adaptation is a major source of antibiotic resistance in bacterial pathogens. Evolution-informed therapy aims to constrain resistance by accounting for bacterial evolvability. Sequential treatments with antibiotics that target different bacterial processes were previously shown to limit adaptation through genetic resistance trade-offs and negative hysteresis. Treatment with homogeneous sets of antibiotics is generally viewed to be disadvantageous, as it should rapidly lead to cross-resistance. We here challenged this assumption by determining the evolutionary response of Pseudomonas aeruginosa to experimental sequential treatments involving both heterogenous and homogeneous antibiotic sets. To our surprise, we found that fast switching between only β-lactam antibiotics resulted in increased extinction of bacterial populations. We demonstrate that extinction is favored by low rates of spontaneous resistance emergence and low levels of spontaneous cross-resistance among the antibiotics in sequence. The uncovered principles may help to guide the optimized use of available antibiotics in highly potent, evolution-informed treatment designs. AU - Batra, Aditi AU - Römhild, Roderich AU - Rousseau, Emilie AU - Franzenburg, Sören AU - Niemann, Stefan AU - Schulenburg, Hinrich ID - 9746 JF - eLife TI - High potency of sequential therapy with only beta-lactam antibiotics VL - 10 ER - TY - JOUR AB - Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10−14 M, allowing an estimate of the number of receptor–ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia. AU - Lee, Jungmin AU - Vernet, Andyna AU - Gruber, Nathalie AU - Kready, Kasia M. AU - Burrill, Devin R. AU - Way, Jeffrey C. AU - Silver, Pamela A. ID - 10363 JF - Protein Engineering, Design and Selection SN - 1741-0126 TI - Rational engineering of an erythropoietin fusion protein to treat hypoxia VL - 34 ER - TY - JOUR AB - Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks (GRNs) remains a major challenge. Here, we use a well-defined synthetic GRN to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one GRN with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit (TU) within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual TUs, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of GRNs. AU - Nagy-Staron, Anna A AU - Tomasek, Kathrin AU - Caruso Carter, Caroline AU - Sonnleitner, Elisabeth AU - Kavcic, Bor AU - Paixão, Tiago AU - Guet, Calin C ID - 9283 JF - eLife KW - Genetics and Molecular Biology SN - 2050-084X TI - Local genetic context shapes the function of a gene regulatory network VL - 10 ER - TY - THES AB - Bacteria-host interactions represent a continuous trade-off between benefit and risk. Thus, the host immune response is faced with a non-trivial problem – accommodate beneficial commensals and remove harmful pathogens. This is especially difficult as molecular patterns, such as lipopolysaccharide or specific surface organelles such as pili, are conserved in both, commensal and pathogenic bacteria. Type 1 pili, tightly regulated by phase variation, are considered an important virulence factor of pathogenic bacteria as they facilitate invasion into host cells. While invasion represents a de facto passive mechanism for pathogens to escape the host immune response, we demonstrate a fundamental role of type 1 pili as active modulators of the innate and adaptive immune response. AU - Tomasek, Kathrin ID - 10307 SN - 2663-337X TI - Pathogenic Escherichia coli hijack the host immune response ER - TY - GEN AB - A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host’s immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on dendritic cells as a previously undescribed binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of pathogenic bacteria to CD14 lead to reduced dendritic cell migration and blunted expression of co-stimulatory molecules, both rate-limiting factors of T cell activation. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn’s disease. AU - Tomasek, Kathrin AU - Leithner, Alexander F AU - Glatzová, Ivana AU - Lukesch, Michael S. AU - Guet, Calin C AU - Sixt, Michael K ID - 10316 T2 - bioRxiv TI - Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14 ER - TY - CHAP AB - Understanding how the activity of membrane receptors and cellular signaling pathways shapes cell behavior is of fundamental interest in basic and applied research. Reengineering receptors to react to light instead of their cognate ligands allows for generating defined signaling inputs with high spatial and temporal precision and facilitates the dissection of complex signaling networks. Here, we describe fundamental considerations in the design of light-regulated receptor tyrosine kinases (Opto-RTKs) and appropriate control experiments. We also introduce methods for transient receptor expression in HEK293 cells, quantitative assessment of signaling activity in reporter gene assays, semiquantitative assessment of (in)activation time courses through Western blot (WB) analysis, and easy to implement light stimulation hardware. AU - Kainrath, Stephanie AU - Janovjak, Harald L ED - Niopek, Dominik ID - 8173 T2 - Photoswitching Proteins TI - Design and application of light-regulated receptor tyrosine kinases VL - 2173 ER - TY - THES AB - In the thesis we focus on the interplay of the biophysics and evolution of gene regulation. We start by addressing how the type of prokaryotic gene regulation – activation and repression – affects spurious binding to DNA, also known as transcriptional crosstalk. We propose that regulatory interference caused by excess regulatory proteins in the dense cellular medium – global crosstalk – could be a factor in determining which type of gene regulatory network is evolutionarily preferred. Next,we use a normative approach in eukaryotic gene regulation to describe minimal non-equilibrium enhancer models that optimize so-called regulatory phenotypes. We find a class of models that differ from standard thermodynamic equilibrium models by a single parameter that notably increases the regulatory performance. Next chapter addresses the question of genotype-phenotype-fitness maps of higher dimensional phenotypes. We show that our biophysically realistic approach allows us to understand how the mechanisms of promoter function constrain genotypephenotype maps, and how they affect the evolutionary trajectories of promoters. In the last chapter we ask whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. Using mathematical modeling, we show that amplifications can tune gene expression in many environments, including those where transcription factor-based schemes are hard to evolve or maintain. AU - Grah, Rok ID - 8155 SN - 2663-337X TI - Gene regulation across scales – how biophysical constraints shape evolution ER - TY - GEN AB - In prokaryotes, thermodynamic models of gene regulation provide a highly quantitative mapping from promoter sequences to gene expression levels that is compatible with in vivo and in vitro bio-physical measurements. Such concordance has not been achieved for models of enhancer function in eukaryotes. In equilibrium models, it is difficult to reconcile the reported short transcription factor (TF) residence times on the DNA with the high specificity of regulation. In non-equilibrium models, progress is difficult due to an explosion in the number of parameters. Here, we navigate this complexity by looking for minimal non-equilibrium enhancer models that yield desired regulatory phenotypes: low TF residence time, high specificity and tunable cooperativity. We find that a single extra parameter, interpretable as the “linking rate” by which bound TFs interact with Mediator components, enables our models to escape equilibrium bounds and access optimal regulatory phenotypes, while remaining consistent with the reported phenomenology and simple enough to be inferred from upcoming experiments. We further find that high specificity in non-equilibrium models is in a tradeoff with gene expression noise, predicting bursty dynamics — an experimentally-observed hallmark of eukaryotic transcription. By drastically reducing the vast parameter space to a much smaller subspace that optimally realizes biological function prior to inference from data, our normative approach holds promise for mathematical models in systems biology. AU - Grah, Rok AU - Zoller, Benjamin AU - Tkačik, Gašper ID - 7675 T2 - bioRxiv TI - Normative models of enhancer function ER - TY - JOUR AB - Genes differ in the frequency at which they are expressed and in the form of regulation used to control their activity. In particular, positive or negative regulation can lead to activation of a gene in response to an external signal. Previous works proposed that the form of regulation of a gene correlates with its frequency of usage: positive regulation when the gene is frequently expressed and negative regulation when infrequently expressed. Such network design means that, in the absence of their regulators, the genes are found in their least required activity state, hence regulatory intervention is often necessary. Due to the multitude of genes and regulators, spurious binding and unbinding events, called “crosstalk”, could occur. To determine how the form of regulation affects the global crosstalk in the network, we used a mathematical model that includes multiple regulators and multiple target genes. We found that crosstalk depends non-monotonically on the availability of regulators. Our analysis showed that excess use of regulation entailed by the formerly suggested network design caused high crosstalk levels in a large part of the parameter space. We therefore considered the opposite ‘idle’ design, where the default unregulated state of genes is their frequently required activity state. We found, that ‘idle’ design minimized the use of regulation and thus minimized crosstalk. In addition, we estimated global crosstalk of S. cerevisiae using transcription factors binding data. We demonstrated that even partial network data could suffice to estimate its global crosstalk, suggesting its applicability to additional organisms. We found that S. cerevisiae estimated crosstalk is lower than that of a random network, suggesting that natural selection reduces crosstalk. In summary, our study highlights a new type of protein production cost which is typically overlooked: that of regulatory interference caused by the presence of excess regulators in the cell. It demonstrates the importance of whole-network descriptions, which could show effects missed by single-gene models. AU - Grah, Rok AU - Friedlander, Tamar ID - 7569 IS - 2 JF - PLOS Computational Biology SN - 1553-7358 TI - The relation between crosstalk and gene regulation form revisited VL - 16 ER - TY - DATA AB - Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions, such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks remains a major challenge. Here, we use a well-defined synthetic gene regulatory network to study how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one gene regulatory network with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Our results demonstrate that changes in local genetic context can place a single transcriptional unit within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual transcriptional units, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of gene regulatory networks. AU - Nagy-Staron, Anna A ID - 8951 KW - Gene regulatory networks KW - Gene expression KW - Escherichia coli KW - Synthetic Biology TI - Sequences of gene regulatory network permutations for the article "Local genetic context shapes the function of a gene regulatory network" ER - TY - DATA AB - Organisms cope with change by employing transcriptional regulators. However, when faced with rare environments, the evolution of transcriptional regulators and their promoters may be too slow. We ask whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. By real-time monitoring of gene copy number mutations in E. coli, we show that gene duplications and amplifications enable adaptation to fluctuating environments by rapidly generating copy number, and hence expression level, polymorphism. This ‘amplification-mediated gene expression tuning’ occurs on timescales similar to canonical gene regulation and can deal with rapid environmental changes. Mathematical modeling shows that amplifications also tune gene expression in stochastic environments where transcription factor-based schemes are hard to evolve or maintain. The fleeting nature of gene amplifications gives rise to a generic population-level mechanism that relies on genetic heterogeneity to rapidly tune expression of any gene, without leaving any genomic signature. AU - Grah, Rok ID - 7383 KW - Matlab scripts KW - analysis of microfluidics KW - mathematical model TI - Matlab scripts for the Paper: Gene Amplification as a Form of Population-Level Gene Expression regulation ER - TY - THES AB - Proteins and their complex dynamic interactions regulate cellular mechanisms from sensing and transducing extracellular signals, to mediating genetic responses, and sustaining or changing cell morphology. To manipulate these protein-protein interactions (PPIs) that govern the behavior and fate of cells, synthetically constructed, genetically encoded tools provide the means to precisely target proteins of interest (POIs), and control their subcellular localization and activity in vitro and in vivo. Ideal synthetic tools react to an orthogonal cue, i.e. a trigger that does not activate any other endogenous process, thereby allowing manipulation of the POI alone. In optogenetics, naturally occurring photosensory domain from plants, algae and bacteria are re-purposed and genetically fused to POIs. Illumination with light of a specific wavelength triggers a conformational change that can mediate PPIs, such as dimerization or oligomerization. By using light as a trigger, these tools can be activated with high spatial and temporal precision, on subcellular and millisecond scales. Chemogenetic tools consist of protein domains that recognize and bind small molecules. By genetic fusion to POIs, these domains can mediate PPIs upon addition of their specific ligands, which are often synthetically designed to provide highly specific interactions and exhibit good bioavailability. Most optogenetic tools to mediate PPIs are based on well-studied photoreceptors responding to red, blue or near-UV light, leaving a striking gap in the green band of the visible light spectrum. Among both optogenetic and chemogenetic tools, there is an abundance of methods to induce PPIs, but tools to disrupt them require UV illumination, rely on covalent linkage and subsequent enzymatic cleavage or initially result in protein clustering of unknown stoichiometry. This work describes how the recently structurally and photochemically characterized green-light responsive cobalamin-binding domains (CBDs) from bacterial transcription factors were re-purposed to function as a green-light responsive optogenetic tool. In contrast to previously engineered optogenetic tools, CBDs do not induce PPI, but rather confer a PPI already upon expression, which can be rapidly disrupted by illumination. This was employed to mimic inhibition of constitutive activity of a growth factor receptor, and successfully implement for cell signalling in mammalian cells and in vivo to rescue development in zebrafish. This work further describes the development and application of a chemically induced de-dimerizer (CDD) based on a recently identified and structurally described bacterial oxyreductase. CDD forms a dimer upon expression in absence of its cofactor, the flavin derivative F420. Safety and of domain expression and ligand exposure are demonstrated in vitro and in vivo in zebrafish. The system is further applied to inhibit cell signalling output from a chimeric receptor upon F420 treatment. CBDs and CDD expand the repertoire of synthetic tools by providing novel mechanisms of mediating PPIs, and by recognizing previously not utilized cues. In the future, they can readily be combined with existing synthetic tools to functionally manipulate PPIs in vitro and in vivo. AU - Kainrath, Stephanie ID - 7680 TI - Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals ER - TY - JOUR AB - Organisms cope with change by taking advantage of transcriptional regulators. However, when faced with rare environments, the evolution of transcriptional regulators and their promoters may be too slow. Here, we investigate whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. Using real-time monitoring of gene-copy-number mutations in Escherichia coli, we show that gene duplications and amplifications enable adaptation to fluctuating environments by rapidly generating copy-number and, therefore, expression-level polymorphisms. This amplification-mediated gene expression tuning (AMGET) occurs on timescales that are similar to canonical gene regulation and can respond to rapid environmental changes. Mathematical modelling shows that amplifications also tune gene expression in stochastic environments in which transcription-factor-based schemes are hard to evolve or maintain. The fleeting nature of gene amplifications gives rise to a generic population-level mechanism that relies on genetic heterogeneity to rapidly tune the expression of any gene, without leaving any genomic signature. AU - Tomanek, Isabella AU - Grah, Rok AU - Lagator, M. AU - Andersson, A. M. C. AU - Bollback, Jonathan P AU - Tkačik, Gašper AU - Guet, Calin C ID - 7652 IS - 4 JF - Nature Ecology & Evolution SN - 2397-334X TI - Gene amplification as a form of population-level gene expression regulation VL - 4 ER - TY - THES AB - Mutations are the raw material of evolution and come in many different flavors. Point mutations change a single letter in the DNA sequence, while copy number mutations like duplications or deletions add or remove many letters of the DNA sequence simultaneously. Each type of mutation exhibits specific properties like its rate of formation and reversal. Gene expression is a fundamental phenotype that can be altered by both, point and copy number mutations. The following thesis is concerned with the dynamics of gene expression evolution and how it is affected by the properties exhibited by point and copy number mutations. Specifically, we are considering i) copy number mutations during adaptation to fluctuating environments and ii) the interaction of copy number and point mutations during adaptation to constant environments.   AU - Tomanek, Isabella ID - 8653 KW - duplication KW - amplification KW - promoter KW - CNV KW - AMGET KW - experimental evolution KW - Escherichia coli SN - 2663-337X TI - The evolution of gene expression by copy number and point mutations ER - TY - JOUR AB - Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells. AU - Chassin, Hélène AU - Müller, Marius AU - Tigges, Marcel AU - Scheller, Leo AU - Lang, Moritz AU - Fussenegger, Martin ID - 6465 IS - 1 JF - Nature Communications TI - A modular degron library for synthetic circuits in mammalian cells VL - 10 ER - TY - JOUR AB - With the recent publication by Silpe and Bassler (2019), considering phage detection of a bacterial quorum-sensing (QS) autoinducer, we now have as many as five examples of phage-associated intercellular communication (Table 1). Each potentially involves ecological inferences by phages as to concentrations of surrounding phage-infected or uninfected bacteria. While the utility of phage detection of bacterial QS molecules may at first glance appear to be straightforward, we suggest in this commentary that the underlying ecological explanation is unlikely to be simple. AU - Igler, Claudia AU - Abedon, Stephen T. ID - 6717 JF - Frontiers in Microbiology TI - Commentary: A host-produced quorum-sensing autoinducer controls a phage lysis-lysogeny decision VL - 10 ER - TY - JOUR AB - Mathematical models have been used successfully at diverse scales of biological organization, ranging from ecology and population dynamics to stochastic reaction events occurring between individual molecules in single cells. Generally, many biological processes unfold across multiple scales, with mutations being the best studied example of how stochasticity at the molecular scale can influence outcomes at the population scale. In many other contexts, however, an analogous link between micro- and macro-scale remains elusive, primarily due to the challenges involved in setting up and analyzing multi-scale models. Here, we employ such a model to investigate how stochasticity propagates from individual biochemical reaction events in the bacterial innate immune system to the ecology of bacteria and bacterial viruses. We show analytically how the dynamics of bacterial populations are shaped by the activities of immunity-conferring enzymes in single cells and how the ecological consequences imply optimal bacterial defense strategies against viruses. Our results suggest that bacterial populations in the presence of viruses can either optimize their initial growth rate or their population size, with the first strategy favoring simple immunity featuring a single restriction modification system and the second strategy favoring complex bacterial innate immunity featuring several simultaneously active restriction modification systems. AU - Ruess, Jakob AU - Pleska, Maros AU - Guet, Calin C AU - Tkačik, Gašper ID - 6784 IS - 7 JF - PLoS Computational Biology TI - Molecular noise of innate immunity shapes bacteria-phage ecologies VL - 15 ER - TY - GEN AU - Ruess, Jakob AU - Pleska, Maros AU - Guet, Calin C AU - Tkačik, Gašper ID - 9786 TI - Supporting text and results ER - TY - CONF AB - The expression of a gene is characterised by its transcription factors and the function processing them. If the transcription factors are not affected by gene products, the regulating function is often represented as a combinational logic circuit, where the outputs (product) are determined by current input values (transcription factors) only, and are hence independent on their relative arrival times. However, the simultaneous arrival of transcription factors (TFs) in genetic circuits is a strong assumption, given that the processes of transcription and translation of a gene into a protein introduce intrinsic time delays and that there is no global synchronisation among the arrival times of different molecular species at molecular targets. In this paper, we construct an experimentally implementable genetic circuit with two inputs and a single output, such that, in presence of small delays in input arrival, the circuit exhibits qualitatively distinct observable phenotypes. In particular, these phenotypes are long lived transients: they all converge to a single value, but so slowly, that they seem stable for an extended time period, longer than typical experiment duration. We used rule-based language to prototype our circuit, and we implemented a search for finding the parameter combinations raising the phenotypes of interest. The behaviour of our prototype circuit has wide implications. First, it suggests that GRNs can exploit event timing to create phenotypes. Second, it opens the possibility that GRNs are using event timing to react to stimuli and memorise events, without explicit feedback in regulation. From the modelling perspective, our prototype circuit demonstrates the critical importance of analysing the transient dynamics at the promoter binding sites of the DNA, before applying rapid equilibrium assumptions. AU - Guet, Calin C AU - Henzinger, Thomas A AU - Igler, Claudia AU - Petrov, Tatjana AU - Sezgin, Ali ID - 7147 SN - 0302-9743 T2 - 17th International Conference on Computational Methods in Systems Biology TI - Transient memory in gene regulation VL - 11773 ER - TY - JOUR AB - Autoregulation is the direct modulation of gene expression by the product of the corresponding gene. Autoregulation of bacterial gene expression has been mostly studied at the transcriptional level, when a protein acts as the cognate transcriptional repressor. A recent study investigating dynamics of the bacterial toxin–antitoxin MazEF system has shown how autoregulation at both the transcriptional and post-transcriptional levels affects the heterogeneity of Escherichia coli populations. Toxin–antitoxin systems hold a crucial but still elusive part in bacterial response to stress. This perspective highlights how these modules can also serve as a great model system for investigating basic concepts in gene regulation. However, as the genomic background and environmental conditions substantially influence toxin activation, it is important to study (auto)regulation of toxin–antitoxin systems in well-defined setups as well as in conditions that resemble the environmental niche. AU - Nikolic, Nela ID - 138 IS - 1 JF - Current Genetics TI - Autoregulation of bacterial gene expression: lessons from the MazEF toxin–antitoxin system VL - 65 ER - TY - JOUR AB - The abelian sandpile serves as a model to study self-organized criticality, a phenomenon occurring in biological, physical and social processes. The identity of the abelian group is a fractal composed of self-similar patches, and its limit is subject of extensive collaborative research. Here, we analyze the evolution of the sandpile identity under harmonic fields of different orders. We show that this evolution corresponds to periodic cycles through the abelian group characterized by the smooth transformation and apparent conservation of the patches constituting the identity. The dynamics induced by second and third order harmonics resemble smooth stretchings, respectively translations, of the identity, while the ones induced by fourth order harmonics resemble magnifications and rotations. Starting with order three, the dynamics pass through extended regions of seemingly random configurations which spontaneously reassemble into accentuated patterns. We show that the space of harmonic functions projects to the extended analogue of the sandpile group, thus providing a set of universal coordinates identifying configurations between different domains. Since the original sandpile group is a subgroup of the extended one, this directly implies that it admits a natural renormalization. Furthermore, we show that the harmonic fields can be induced by simple Markov processes, and that the corresponding stochastic dynamics show remarkable robustness over hundreds of periods. Finally, we encode information into seemingly random configurations, and decode this information with an algorithm requiring minimal prior knowledge. Our results suggest that harmonic fields might split the sandpile group into sub-sets showing different critical coefficients, and that it might be possible to extend the fractal structure of the identity beyond the boundaries of its domain. AU - Lang, Moritz AU - Shkolnikov, Mikhail ID - 196 IS - 8 JF - Proceedings of the National Academy of Sciences TI - Harmonic dynamics of the Abelian sandpile VL - 116 ER - TY - DATA AB - Organisms cope with change by employing transcriptional regulators. However, when faced with rare environments, the evolution of transcriptional regulators and their promoters may be too slow. We ask whether the intrinsic instability of gene duplication and amplification provides a generic alternative to canonical gene regulation. By real-time monitoring of gene copy number mutations in E. coli, we show that gene duplications and amplifications enable adaptation to fluctuating environments by rapidly generating copy number, and hence expression level, polymorphism. This ‘amplification-mediated gene expression tuning’ occurs on timescales similar to canonical gene regulation and can deal with rapid environmental changes. Mathematical modeling shows that amplifications also tune gene expression in stochastic environments where transcription factor-based schemes are hard to evolve or maintain. The fleeting nature of gene amplifications gives rise to a generic population-level mechanism that relies on genetic heterogeneity to rapidly tune expression of any gene, without leaving any genomic signature. AU - Tomanek, Isabella ID - 7016 KW - Escherichia coli KW - gene amplification KW - galactose KW - DOG KW - experimental evolution KW - Illumina sequence data KW - FACS data KW - microfluidics data TI - Data for the paper "Gene amplification as a form of population-level gene expression regulation" ER - TY - THES AB - Decades of studies have revealed the mechanisms of gene regulation in molecular detail. We make use of such well-described regulatory systems to explore how the molecular mechanisms of protein-protein and protein-DNA interactions shape the dynamics and evolution of gene regulation. i) We uncover how the biophysics of protein-DNA binding determines the potential of regulatory networks to evolve and adapt, which can be captured using a simple mathematical model. ii) The evolution of regulatory connections can lead to a significant amount of crosstalk between binding proteins. We explore the effect of crosstalk on gene expression from a target promoter, which seems to be modulated through binding competition at non-specific DNA sites. iii) We investigate how the very same biophysical characteristics as in i) can generate significant fitness costs for cells through global crosstalk, meaning non-specific DNA binding across the genomic background. iv) Binding competition between proteins at a target promoter is a prevailing regulatory feature due to the prevalence of co-regulation at bacterial promoters. However, the dynamics of these systems are not always straightforward to determine even if the molecular mechanisms of regulation are known. A detailed model of the biophysical interactions reveals that interference between the regulatory proteins can constitute a new, generic form of system memory that records the history of the input signals at the promoter. We demonstrate how the biophysics of protein-DNA binding can be harnessed to investigate the principles that shape and ultimately limit cellular gene regulation. These results provide a basis for studies of higher-level functionality, which arises from the underlying regulation. AU - Igler, Claudia ID - 6371 KW - gene regulation KW - biophysics KW - transcription factor binding KW - bacteria SN - 2663-337X TI - On the nature of gene regulatory design - The biophysics of transcription factor binding shapes gene regulation ER - TY - JOUR AB - The hanging-drop network (HDN) is a technology platform based on a completely open microfluidic network at the bottom of an inverted, surface-patterned substrate. The platform is predominantly used for the formation, culturing, and interaction of self-assembled spherical microtissues (spheroids) under precisely controlled flow conditions. Here, we describe design, fabrication, and operation of microfluidic hanging-drop networks. AU - Misun, Patrick AU - Birchler, Axel AU - Lang, Moritz AU - Hierlemann, Andreas AU - Frey, Olivier ID - 305 JF - Methods in Molecular Biology TI - Fabrication and operation of microfluidic hanging drop networks VL - 1771 ER - TY - JOUR AB - Escaping local optima is one of the major obstacles to function optimisation. Using the metaphor of a fitness landscape, local optima correspond to hills separated by fitness valleys that have to be overcome. We define a class of fitness valleys of tunable difficulty by considering their length, representing the Hamming path between the two optima and their depth, the drop in fitness. For this function class we present a runtime comparison between stochastic search algorithms using different search strategies. The (1+1) EA is a simple and well-studied evolutionary algorithm that has to jump across the valley to a point of higher fitness because it does not accept worsening moves (elitism). In contrast, the Metropolis algorithm and the Strong Selection Weak Mutation (SSWM) algorithm, a famous process in population genetics, are both able to cross the fitness valley by accepting worsening moves. We show that the runtime of the (1+1) EA depends critically on the length of the valley while the runtimes of the non-elitist algorithms depend crucially on the depth of the valley. Moreover, we show that both SSWM and Metropolis can also efficiently optimise a rugged function consisting of consecutive valleys. AU - Oliveto, Pietro AU - Paixao, Tiago AU - Pérez Heredia, Jorge AU - Sudholt, Dirk AU - Trubenova, Barbora ID - 723 IS - 5 JF - Algorithmica TI - How to escape local optima in black box optimisation when non elitism outperforms elitism VL - 80 ER - TY - JOUR AB - Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg2+ and Ca2+, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry. AU - Tomasek, Kathrin AU - Bergmiller, Tobias AU - Guet, Calin C ID - 503 JF - Journal of Biotechnology TI - Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains VL - 268 ER - TY - JOUR AB - In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIRis maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIRin these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility—a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria—their “existence conditions.”. AU - Chaudhry, Waqas AU - Pleska, Maros AU - Shah, Nilang AU - Weiss, Howard AU - Mccall, Ingrid AU - Meyer, Justin AU - Gupta, Animesh AU - Guet, Calin C AU - Levin, Bruce ID - 82 IS - 8 JF - PLoS Biology TI - Leaky resistance and the conditions for the existence of lytic bacteriophage VL - 16 ER - TY - GEN AU - Chaudhry, Waqas AU - Pleska, Maros AU - Shah, Nilang AU - Weiss, Howard AU - Mccall, Ingrid AU - Meyer, Justin AU - Gupta, Animesh AU - Guet, Calin C AU - Levin, Bruce ID - 9810 TI - Numerical data used in figures ER - TY - JOUR AB - Temperate bacteriophages integrate in bacterial genomes as prophages and represent an important source of genetic variation for bacterial evolution, frequently transmitting fitness-augmenting genes such as toxins responsible for virulence of major pathogens. However, only a fraction of bacteriophage infections are lysogenic and lead to prophage acquisition, whereas the majority are lytic and kill the infected bacteria. Unless able to discriminate lytic from lysogenic infections, mechanisms of immunity to bacteriophages are expected to act as a double-edged sword and increase the odds of survival at the cost of depriving bacteria of potentially beneficial prophages. We show that although restriction-modification systems as mechanisms of innate immunity prevent both lytic and lysogenic infections indiscriminately in individual bacteria, they increase the number of prophage-acquiring individuals at the population level. We find that this counterintuitive result is a consequence of phage-host population dynamics, in which restriction-modification systems delay infection onset until bacteria reach densities at which the probability of lysogeny increases. These results underscore the importance of population-level dynamics as a key factor modulating costs and benefits of immunity to temperate bacteriophages AU - Pleska, Maros AU - Lang, Moritz AU - Refardt, Dominik AU - Levin, Bruce AU - Guet, Calin C ID - 457 IS - 2 JF - Nature Ecology and Evolution TI - Phage-host population dynamics promotes prophage acquisition in bacteria with innate immunity VL - 2 ER - TY - JOUR AB - G-protein-coupled receptors (GPCRs) form the largest receptor family, relay environmental stimuli to changes in cell behavior and represent prime drug targets. Many GPCRs are classified as orphan receptors because of the limited knowledge on their ligands and coupling to cellular signaling machineries. Here, we engineer a library of 63 chimeric receptors that contain the signaling domains of human orphan and understudied GPCRs functionally linked to the light-sensing domain of rhodopsin. Upon stimulation with visible light, we identify activation of canonical cell signaling pathways, including cAMP-, Ca2+-, MAPK/ERK-, and Rho-dependent pathways, downstream of the engineered receptors. For the human pseudogene GPR33, we resurrect a signaling function that supports its hypothesized role as a pathogen entry site. These results demonstrate that substituting unknown chemical activators with a light switch can reveal information about protein function and provide an optically controlled protein library for exploring the physiology and therapeutic potential of understudied GPCRs. AU - Morri, Maurizio AU - Sanchez-Romero, Inmaculada AU - Tichy, Alexandra-Madelaine AU - Kainrath, Stephanie AU - Gerrard, Elliot J. AU - Hirschfeld, Priscila AU - Schwarz, Jan AU - Janovjak, Harald L ID - 5984 IS - 1 JF - Nature Communications SN - 2041-1723 TI - Optical functionalization of human class A orphan G-protein-coupled receptors VL - 9 ER - TY - JOUR AB - Bacteria regulate genes to survive antibiotic stress, but regulation can be far from perfect. When regulation is not optimal, mutations that change gene expression can contribute to antibiotic resistance. It is not systematically understood to what extent natural gene regulation is or is not optimal for distinct antibiotics, and how changes in expression of specific genes quantitatively affect antibiotic resistance. Here we discover a simple quantitative relation between fitness, gene expression, and antibiotic potency, which rationalizes our observation that a multitude of genes and even innate antibiotic defense mechanisms have expression that is critically nonoptimal under antibiotic treatment. First, we developed a pooled-strain drug-diffusion assay and screened Escherichia coli overexpression and knockout libraries, finding that resistance to a range of 31 antibiotics could result from changing expression of a large and functionally diverse set of genes, in a primarily but not exclusively drug-specific manner. Second, by synthetically controlling the expression of single-drug and multidrug resistance genes, we observed that their fitness-expression functions changed dramatically under antibiotic treatment in accordance with a log-sensitivity relation. Thus, because many genes are nonoptimally expressed under antibiotic treatment, many regulatory mutations can contribute to resistance by altering expression and by activating latent defenses. AU - Palmer, Adam AU - Chait, Remy P AU - Kishony, Roy ID - 19 IS - 11 JF - Molecular Biology and Evolution SN - 0737-4038 TI - Nonoptimal gene expression creates latent potential for antibiotic resistance VL - 35 ER - TY - JOUR AB - The MazF toxin sequence-specifically cleaves single-stranded RNA upon various stressful conditions, and it is activated as a part of the mazEF toxin–antitoxin module in Escherichia coli. Although autoregulation of mazEF expression through the MazE antitoxin-dependent transcriptional repression has been biochemically characterized, less is known about post-transcriptional autoregulation, as well as how both of these autoregulatory features affect growth of single cells during conditions that promote MazF production. Here, we demonstrate post-transcriptional autoregulation of mazF expression dynamics by MazF cleaving its own transcript. Single-cell analyses of bacterial populations during ectopic MazF production indicated that two-level autoregulation of mazEF expression influences cell-to-cell growth rate heterogeneity. The increase in growth rate heterogeneity is governed by the MazE antitoxin, and tuned by the MazF-dependent mazF mRNA cleavage. Also, both autoregulatory features grant rapid exit from the stress caused by mazF overexpression. Time-lapse microscopy revealed that MazF-mediated cleavage of mazF mRNA leads to increased temporal variability in length of individual cells during ectopic mazF overexpression, as explained by a stochastic model indicating that mazEF mRNA cleavage underlies temporal fluctuations in MazF levels during stress. AU - Nikolic, Nela AU - Bergmiller, Tobias AU - Vandervelde, Alexandra AU - Albanese, Tanino AU - Gelens, Lendert AU - Moll, Isabella ID - 438 IS - 6 JF - Nucleic Acids Research TI - Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations VL - 46 ER - TY - DATA AB - Nela Nikolic, Tobias Bergmiller, Alexandra Vandervelde, Tanino G. Albanese, Lendert Gelens, and Isabella Moll (2018) “Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations” Nucleic Acids Research, doi: 10.15479/AT:ISTA:74; microscopy experiments by Tobias Bergmiller; image and data analysis by Nela Nikolic. AU - Bergmiller, Tobias AU - Nikolic, Nela ID - 5569 KW - microscopy KW - microfluidics TI - Time-lapse microscopy data ER - TY - JOUR AB - Which properties of metabolic networks can be derived solely from stoichiometry? Predictive results have been obtained by flux balance analysis (FBA), by postulating that cells set metabolic fluxes to maximize growth rate. Here we consider a generalization of FBA to single-cell level using maximum entropy modeling, which we extend and test experimentally. Specifically, we define for Escherichia coli metabolism a flux distribution that yields the experimental growth rate: the model, containing FBA as a limit, provides a better match to measured fluxes and it makes a wide range of predictions: on flux variability, regulation, and correlations; on the relative importance of stoichiometry vs. optimization; on scaling relations for growth rate distributions. We validate the latter here with single-cell data at different sub-inhibitory antibiotic concentrations. The model quantifies growth optimization as emerging from the interplay of competitive dynamics in the population and regulation of metabolism at the level of single cells. AU - De Martino, Daniele AU - Mc, Andersson Anna AU - Bergmiller, Tobias AU - Guet, Calin C AU - Tkacik, Gasper ID - 161 IS - 1 JF - Nature Communications TI - Statistical mechanics for metabolic networks during steady state growth VL - 9 ER - TY - THES AB - Expression of genes is a fundamental molecular phenotype that is subject to evolution by different types of mutations. Both the rate and the effect of mutations may depend on the DNA sequence context of a particular gene or a particular promoter sequence. In this thesis I investigate the nature of this dependence using simple genetic systems in Escherichia coli. With these systems I explore the evolution of constitutive gene expression from random starting sequences at different loci on the chromosome and at different locations in sequence space. First, I dissect chromosomal neighborhood effects that underlie locus-dependent differences in the potential of a gene under selection to become more highly expressed. Next, I find that the effects of point mutations in promoter sequences are dependent on sequence context, and that an existing energy matrix model performs poorly in predicting relative expression of unrelated sequences. Finally, I show that a substantial fraction of random sequences contain functional promoters and I present an extended thermodynamic model that predicts promoter strength in full sequence space. Taken together, these results provide new insights and guides on how to integrate information on sequence context to improve our qualitative and quantitative understanding of bacterial gene expression, with implications for rapid evolution of drug resistance, de novo evolution of genes, and horizontal gene transfer. AU - Steinrück, Magdalena ID - 26 SN - 2663-337X TI - The influence of sequence context on the evolution of bacterial gene expression ER - TY - JOUR AB - Gene regulatory networks evolve through rewiring of individual components—that is, through changes in regulatory connections. However, the mechanistic basis of regulatory rewiring is poorly understood. Using a canonical gene regulatory system, we quantify the properties of transcription factors that determine the evolutionary potential for rewiring of regulatory connections: robustness, tunability and evolvability. In vivo repression measurements of two repressors at mutated operator sites reveal their contrasting evolutionary potential: while robustness and evolvability were positively correlated, both were in trade-off with tunability. Epistatic interactions between adjacent operators alleviated this trade-off. A thermodynamic model explains how the differences in robustness, tunability and evolvability arise from biophysical characteristics of repressor–DNA binding. The model also uncovers that the energy matrix, which describes how mutations affect repressor–DNA binding, encodes crucial information about the evolutionary potential of a repressor. The biophysical determinants of evolutionary potential for regulatory rewiring constitute a mechanistic framework for understanding network evolution. AU - Igler, Claudia AU - Lagator, Mato AU - Tkacik, Gasper AU - Bollback, Jonathan P AU - Guet, Calin C ID - 67 IS - 10 JF - Nature Ecology and Evolution TI - Evolutionary potential of transcription factors for gene regulatory rewiring VL - 2 ER - TY - DATA AB - Mean repression values and standard error of the mean are given for all operator mutant libraries. AU - Igler, Claudia AU - Lagator, Mato AU - Tkacik, Gasper AU - Bollback, Jonathan P AU - Guet, Calin C ID - 5585 TI - Data for the paper Evolutionary potential of transcription factors for gene regulatory rewiring ER - TY - JOUR AB - Optogenetik und Photopharmakologie ermöglichen präzise räumliche und zeitliche Kontrolle von Proteinwechselwirkung und -funktion in Zellen und Tieren. Optogenetische Methoden, die auf grünes Licht ansprechen und zum Trennen von Proteinkomplexen geeignet sind, sind nichtweitläufig verfügbar, würden jedoch mehrfarbige Experimente zur Beantwortung von biologischen Fragestellungen ermöglichen. Hier demonstrieren wir die Verwendung von Cobalamin(Vitamin B12)-bindenden Domänen von bakteriellen CarH-Transkriptionsfaktoren zur Grünlicht-induzierten Dissoziation von Rezeptoren. Fusioniert mit dem Fibroblasten-W achstumsfaktor-Rezeptor 1 führten diese im Dunkeln in kultivierten Zellen zu Signalaktivität durch Oligomerisierung, welche durch Beleuchten umgehend aufgehoben wurde. In Zebrafischembryonen, die einen derartigen Rezeptor exprimieren, ermöglichte grünes Licht die Kontrolle über abnormale Signalaktivität während der Embryonalentwicklung. AU - Kainrath, Stephanie AU - Stadler, Manuela AU - Gschaider-Reichhart, Eva AU - Distel, Martin AU - Janovjak, Harald L ID - 538 IS - 16 JF - Angewandte Chemie TI - Grünlicht-induzierte Rezeptorinaktivierung durch Cobalamin-bindende Domänen VL - 129 ER -