TY - JOUR AB - The rising prevalence of antibiotic resistant bacteria is an increasingly serious public health challenge. To address this problem, recent work ranging from clinical studies to theoretical modeling has provided valuable insights into the mechanisms of resistance, its emergence and spread, and ways to counteract it. A deeper understanding of the underlying dynamics of resistance evolution will require a combination of experimental and theoretical expertise from different disciplines and new technology for studying evolution in the laboratory. Here, we review recent advances in the quantitative understanding of the mechanisms and evolution of antibiotic resistance. We focus on key theoretical concepts and new technology that enables well-controlled experiments. We further highlight key challenges that can be met in the near future to ultimately develop effective strategies for combating resistance. AU - Lukacisinova, Marta AU - Bollenbach, Mark Tobias ID - 1027 JF - Current Opinion in Biotechnology TI - Toward a quantitative understanding of antibiotic resistance evolution VL - 46 ER - TY - JOUR AB - Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue. AU - Schwarz, Jan AU - Bierbaum, Veronika AU - Merrin, Jack AU - Frank, Tino AU - Hauschild, Robert AU - Bollenbach, Mark Tobias AU - Tay, Savaş AU - Sixt, Michael K AU - Mehling, Matthias ID - 1154 JF - Scientific Reports TI - A microfluidic device for measuring cell migration towards substrate bound and soluble chemokine gradients VL - 6 ER - TY - JOUR AB - Investigating the physiology of cyanobacteria cultured under a diel light regime is relevant for a better understanding of the resulting growth characteristics and for specific biotechnological applications that are foreseen for these photosynthetic organisms. Here, we present the results of a multiomics study of the model cyanobacterium Synechocystis sp. strain PCC 6803, cultured in a lab-scale photobioreactor in physiological conditions relevant for large-scale culturing. The culture was sparged withN2 andCO2, leading to an anoxic environment during the dark period. Growth followed the availability of light. Metabolite analysis performed with 1Hnuclear magnetic resonance analysis showed that amino acids involved in nitrogen and sulfur assimilation showed elevated levels in the light. Most protein levels, analyzed through mass spectrometry, remained rather stable. However, several high-light-response proteins and stress-response proteins showed distinct changes at the onset of the light period. Microarray-based transcript analysis found common patterns of~56% of the transcriptome following the diel regime. These oscillating transcripts could be grouped coarsely into genes that were upregulated and downregulated in the dark period. The accumulated glycogen was degraded in the anaerobic environment in the dark. A small part was degraded gradually, reflecting basic maintenance requirements of the cells in darkness. Surprisingly, the largest part was degraded rapidly in a short time span at the end of the dark period. This degradation could allow rapid formation of metabolic intermediates at the end of the dark period, preparing the cells for the resumption of growth at the start of the light period. AU - Angermayr, Andreas AU - Van Alphen, Pascal AU - Hasdemir, Dicle AU - Kramer, Gertjan AU - Iqbal, Muzamal AU - Van Grondelle, Wilmar AU - Hoefsloot, Huub AU - Choi, Younghae AU - Hellingwerf, Klaas ID - 1218 IS - 14 JF - Applied and Environmental Microbiology TI - Culturing synechocystis sp. Strain pcc 6803 with N2 and CO2 in a diel regime reveals multiphase glycogen dynamics with low maintenance costs VL - 82 ER - TY - JOUR AB - Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings. AU - Qi, Qin AU - Toll Riera, Macarena AU - Heilbron, Karl AU - Preston, Gail AU - Maclean, R Craig ID - 1552 IS - 1822 JF - Proceedings of the Royal Society of London Series B Biological Sciences TI - The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa VL - 283 ER - TY - DATA AB - MATLAB code and processed datasets available for reproducing the results in: Lukačišin, M.*, Landon, M.*, Jajoo, R*. (2016) Sequence-Specific Thermodynamic Properties of Nucleic Acids Influence Both Transcriptional Pausing and Backtracking in Yeast. *equal contributions AU - Lukacisin, Martin AU - Landon, Matthieu AU - Jajoo, Rishi ID - 5556 KW - transcription KW - pausing KW - backtracking KW - polymerase KW - RNA KW - NET-seq KW - nucleosome KW - basepairing TI - MATLAB analysis code for 'Sequence-Specific Thermodynamic Properties of Nucleic Acids Influence Both Transcriptional Pausing and Backtracking in Yeast' ER - TY - JOUR AB - Epistatic interactions can frustrate and shape evolutionary change. Indeed, phenotypes may fail to evolve when essential mutations are only accessible through positive selection if they are fixed simultaneously. How environmental variability affects such constraints is poorly understood. Here, we studied genetic constraints in fixed and fluctuating environments using the Escherichia coli lac operon as a model system for genotype-environment interactions. We found that, in different fixed environments, all trajectories that were reconstructed by applying point mutations within the transcription factor-operator interface became trapped at suboptima, where no additional improvements were possible. Paradoxically, repeated switching between these same environments allows unconstrained adaptation by continuous improvements. This evolutionary mode is explained by pervasive cross-environmental tradeoffs that reposition the peaks in such a way that trapped genotypes can repeatedly climb ascending slopes and hence, escape adaptive stasis. Using a Markov approach, we developed a mathematical framework to quantify the landscape-crossing rates and show that this ratchet-like adaptive mechanism is robust in a wide spectrum of fluctuating environments. Overall, this study shows that genetic constraints can be overcome by environmental change and that crossenvironmental tradeoffs do not necessarily impede but also, can facilitate adaptive evolution. Because tradeoffs and environmental variability are ubiquitous in nature, we speculate this evolutionary mode to be of general relevance. AU - De Vos, Marjon AU - Dawid, Alexandre AU - Šunderlíková, Vanda AU - Tans, Sander ID - 1571 IS - 48 JF - PNAS TI - Breaking evolutionary constraint with a tradeoff ratchet VL - 112 ER - TY - JOUR AB - In animal embryos, morphogen gradients determine tissue patterning and morphogenesis. Shyer et al. provide evidence that, during vertebrate gut formation, tissue folding generates graded activity of signals required for subsequent steps of gut growth and differentiation, thereby revealing an intriguing link between tissue morphogenesis and morphogen gradient formation. AU - Bollenbach, Mark Tobias AU - Heisenberg, Carl-Philipp J ID - 1581 IS - 3 JF - Cell TI - Gradients are shaping up VL - 161 ER - TY - JOUR AB - Through metabolic engineering cyanobacteria can be employed in biotechnology. Combining the capacity for oxygenic photosynthesis and carbon fixation with an engineered metabolic pathway allows carbon-based product formation from CO2, light, and water directly. Such cyanobacterial 'cell factories' are constructed to produce biofuels, bioplastics, and commodity chemicals. Efforts of metabolic engineers and synthetic biologists allow the modification of the intermediary metabolism at various branching points, expanding the product range. The new biosynthesis routes 'tap' the metabolism ever more efficiently, particularly through the engineering of driving forces and utilization of cofactors generated during the light reactions of photosynthesis, resulting in higher product titers. High rates of carbon rechanneling ultimately allow an almost-complete allocation of fixed carbon to product above biomass. AU - Angermayr, Andreas AU - Gorchs, Aleix AU - Hellingwerf, Klaas ID - 1586 IS - 6 JF - Trends in Biotechnology TI - Metabolic engineering of cyanobacteria for the synthesis of commodity products VL - 33 ER - TY - JOUR AB - Background Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. Results We present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for l-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive l-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells. Conclusions The workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology. AU - Hammar, Petter AU - Angermayr, Andreas AU - Sjostrom, Staffan AU - Van Der Meer, Josefin AU - Hellingwerf, Klaas AU - Hudson, Elton AU - Joensson, Hakaan ID - 1623 IS - 1 JF - Biotechnology for Biofuels TI - Single-cell screening of photosynthetic growth and lactate production by cyanobacteria VL - 8 ER - TY - JOUR AB - Combining antibiotics is a promising strategy for increasing treatment efficacy and for controlling resistance evolution. When drugs are combined, their effects on cells may be amplified or weakened, that is the drugs may show synergistic or antagonistic interactions. Recent work revealed the underlying mechanisms of such drug interactions by elucidating the drugs'; joint effects on cell physiology. Moreover, new treatment strategies that use drug combinations to exploit evolutionary tradeoffs were shown to affect the rate of resistance evolution in predictable ways. High throughput studies have further identified drug candidates based on their interactions with established antibiotics and general principles that enable the prediction of drug interactions were suggested. Overall, the conceptual and technical foundation for the rational design of potent drug combinations is rapidly developing. AU - Bollenbach, Mark Tobias ID - 1810 JF - Current Opinion in Microbiology TI - Antimicrobial interactions: Mechanisms and implications for drug discovery and resistance evolution VL - 27 ER - TY - JOUR AB - Abstract Drug combinations are increasingly important in disease treatments, for combating drug resistance, and for elucidating fundamental relationships in cell physiology. When drugs are combined, their individual effects on cells may be amplified or weakened. Such drug interactions are crucial for treatment efficacy, but their underlying mechanisms remain largely unknown. To uncover the causes of drug interactions, we developed a systematic approach based on precise quantification of the individual and joint effects of antibiotics on growth of genome-wide Escherichia coli gene deletion strains. We found that drug interactions between antibiotics representing the main modes of action are highly robust to genetic perturbation. This robustness is encapsulated in a general principle of bacterial growth, which enables the quantitative prediction of mutant growth rates under drug combinations. Rare violations of this principle exposed recurring cellular functions controlling drug interactions. In particular, we found that polysaccharide and ATP synthesis control multiple drug interactions with previously unexplained mechanisms, and small molecule adjuvants targeting these functions synthetically reshape drug interactions in predictable ways. These results provide a new conceptual framework for the design of multidrug combinations and suggest that there are universal mechanisms at the heart of most drug interactions. Synopsis A general principle of bacterial growth enables the prediction of mutant growth rates under drug combinations. Rare violations of this principle expose cellular functions that control drug interactions and can be targeted by small molecules to alter drug interactions in predictable ways. Drug interactions between antibiotics are highly robust to genetic perturbations. A general principle of bacterial growth enables the prediction of mutant growth rates under drug combinations. Rare violations of this principle expose cellular functions that control drug interactions. Diverse drug interactions are controlled by recurring cellular functions, including LPS synthesis and ATP synthesis. A general principle of bacterial growth enables the prediction of mutant growth rates under drug combinations. Rare violations of this principle expose cellular functions that control drug interactions and can be targeted by small molecules to alter drug interactions in predictable ways. AU - Chevereau, Guillaume AU - Bollenbach, Mark Tobias ID - 1823 IS - 4 JF - Molecular Systems Biology TI - Systematic discovery of drug interaction mechanisms VL - 11 ER - TY - GEN AU - Chevereau, Guillaume AU - Lukacisinova, Marta AU - Batur, Tugce AU - Guvenek, Aysegul AU - Ayhan, Dilay Hazal AU - Toprak, Erdal AU - Bollenbach, Mark Tobias ID - 9711 TI - Excel file containing the raw data for all figures ER - TY - GEN AU - Chevereau, Guillaume AU - Lukacisinova, Marta AU - Batur, Tugce AU - Guvenek, Aysegul AU - Ayhan, Dilay Hazal AU - Toprak, Erdal AU - Bollenbach, Mark Tobias ID - 9765 TI - Gene ontology enrichment analysis for the most sensitive gene deletion strains for all drugs ER - TY - JOUR AB - The Auxin Binding Protein1 (ABP1) has been identified based on its ability to bind auxin with high affinity and studied for a long time as a prime candidate for the extracellular auxin receptor responsible for mediating in particular the fast non-transcriptional auxin responses. However, the contradiction between the embryo-lethal phenotypes of the originally described Arabidopsis T-DNA insertional knock-out alleles (abp1-1 and abp1-1s) and the wild type-like phenotypes of other recently described loss-of-function alleles (abp1-c1 and abp1-TD1) questions the biological importance of ABP1 and relevance of the previous genetic studies. Here we show that there is no hidden copy of the ABP1 gene in the Arabidopsis genome but the embryo-lethal phenotypes of abp1-1 and abp1-1s alleles are very similar to the knock-out phenotypes of the neighboring gene, BELAYA SMERT (BSM). Furthermore, the allelic complementation test between bsm and abp1 alleles shows that the embryo-lethality in the abp1-1 and abp1-1s alleles is caused by the off-target disruption of the BSM locus by the T-DNA insertions. This clarifies the controversy of different phenotypes among published abp1 knock-out alleles and asks for reflections on the developmental role of ABP1. AU - Michalko, Jaroslav AU - Dravecka, Marta AU - Bollenbach, Tobias AU - Friml, Jirí ID - 1509 JF - F1000 Research TI - Embryo-lethal phenotypes in early abp1 mutants are due to disruption of the neighboring BSM gene VL - 4 ER - TY - JOUR AB - The emergence of drug resistant pathogens is a serious public health problem. It is a long-standing goal to predict rates of resistance evolution and design optimal treatment strategies accordingly. To this end, it is crucial to reveal the underlying causes of drug-specific differences in the evolutionary dynamics leading to resistance. However, it remains largely unknown why the rates of resistance evolution via spontaneous mutations and the diversity of mutational paths vary substantially between drugs. Here we comprehensively quantify the distribution of fitness effects (DFE) of mutations, a key determinant of evolutionary dynamics, in the presence of eight antibiotics representing the main modes of action. Using precise high-throughput fitness measurements for genome-wide Escherichia coli gene deletion strains, we find that the width of the DFE varies dramatically between antibiotics and, contrary to conventional wisdom, for some drugs the DFE width is lower than in the absence of stress. We show that this previously underappreciated divergence in DFE width among antibiotics is largely caused by their distinct drug-specific dose-response characteristics. Unlike the DFE, the magnitude of the changes in tolerated drug concentration resulting from genome-wide mutations is similar for most drugs but exceptionally small for the antibiotic nitrofurantoin, i.e., mutations generally have considerably smaller resistance effects for nitrofurantoin than for other drugs. A population genetics model predicts that resistance evolution for drugs with this property is severely limited and confined to reproducible mutational paths. We tested this prediction in laboratory evolution experiments using the “morbidostat”, a device for evolving bacteria in well-controlled drug environments. Nitrofurantoin resistance indeed evolved extremely slowly via reproducible mutations—an almost paradoxical behavior since this drug causes DNA damage and increases the mutation rate. Overall, we identified novel quantitative characteristics of the evolutionary landscape that provide the conceptual foundation for predicting the dynamics of drug resistance evolution. AU - Chevereau, Guillaume AU - Dravecka, Marta AU - Batur, Tugce AU - Guvenek, Aysegul AU - Ayhan, Dilay AU - Toprak, Erdal AU - Bollenbach, Mark Tobias ID - 1619 IS - 11 JF - PLoS Biology TI - Quantifying the determinants of evolutionary dynamics leading to drug resistance VL - 13 ER - TY - JOUR AB - Development requires tissue growth as well as cell diversification. To address how these processes are coordinated, we analyzed the development of molecularly distinct domains of neural progenitors in the mouse and chick neural tube. We show that during development, these domains undergo changes in size that do not scale with changes in overall tissue size. Our data show that domain proportions are first established by opposing morphogen gradients and subsequently controlled by domain-specific regulation of differentiation rate but not differences in proliferation rate. Regulation of differentiation rate is key to maintaining domain proportions while accommodating both intra- and interspecies variations in size. Thus, the sequential control of progenitor specification and differentiation elaborates pattern without requiring that signaling gradients grow as tissues expand. AU - Kicheva, Anna AU - Bollenbach, Mark Tobias AU - Ribeiro, Ana AU - Pérez Valle, Helena AU - Lovell Badge, Robin AU - Episkopou, Vasso AU - Briscoe, James ID - 2040 IS - 6204 JF - Science TI - Coordination of progenitor specification and growth in mouse and chick spinal cord VL - 345 ER - TY - JOUR AB - In this issue of Chemistry & Biology, Cokol and colleagues report a systematic study of drug interactions between antifungal compounds. Suppressive drug interactions occur more frequently than previously realized and come in different flavors with interesting implications. AU - De Vos, Marjon AU - Bollenbach, Mark Tobias ID - 2220 IS - 4 JF - Chemistry and Biology SN - 10745521 TI - Suppressive drug interactions between antifungals VL - 21 ER - TY - JOUR AB - Antibiotics affect bacterial cell physiology at many levels. Rather than just compensating for the direct cellular defects caused by the drug, bacteria respond to antibiotics by changing their morphology, macromolecular composition, metabolism, gene expression and possibly even their mutation rate. Inevitably, these processes affect each other, resulting in a complex response with changes in the expression of numerous genes. Genome‐wide approaches can thus help in gaining a comprehensive understanding of bacterial responses to antibiotics. In addition, a combination of experimental and theoretical approaches is needed for identifying general principles that underlie these responses. Here, we review recent progress in our understanding of bacterial responses to antibiotics and their combinations, focusing on effects at the levels of growth rate and gene expression. We concentrate on studies performed in controlled laboratory conditions, which combine promising experimental techniques with quantitative data analysis and mathematical modeling. While these basic research approaches are not immediately applicable in the clinic, uncovering the principles and mechanisms underlying bacterial responses to antibiotics may, in the long term, contribute to the development of new treatment strategies to cope with and prevent the rise of resistant pathogenic bacteria. AU - Mitosch, Karin AU - Bollenbach, Tobias ID - 2001 IS - 6 JF - Environmental Microbiology Reports TI - Bacterial responses to antibiotics and their combinations VL - 6 ER - TY - JOUR AB - The epistatic interactions that underlie evolutionary constraint have mainly been studied for constant external conditions. However, environmental changes may modulate epistasis and hence affect genetic constraints. Here we investigate genetic constraints in the adaptive evolution of a novel regulatory function in variable environments, using the lac repressor, LacI, as a model system. We have systematically reconstructed mutational trajectories from wild type LacI to three different variants that each exhibit an inverse response to the inducing ligand IPTG, and analyzed the higher-order interactions between genetic and environmental changes. We find epistasis to depend strongly on the environment. As a result, mutational steps essential to inversion but inaccessible by positive selection in one environment, become accessible in another. We present a graphical method to analyze the observed complex higher-order interactions between multiple mutations and environmental change, and show how the interactions can be explained by a combination of mutational effects on allostery and thermodynamic stability. This dependency of genetic constraint on the environment should fundamentally affect evolutionary dynamics and affects the interpretation of phylogenetic data. AU - De Vos, Marjon AU - Poelwijk, Frank AU - Battich, Nico AU - Ndika, Joseph AU - Tans, Sander ID - 2810 IS - 6 JF - PLoS Genetics TI - Environmental dependence of genetic constraint VL - 9 ER - TY - JOUR AB - Morphogen gradients regulate the patterning and growth of many tissues, hence a key question is how they are established and maintained during development. Theoretical descriptions have helped to explain how gradient shape is controlled by the rates of morphogen production, spreading and degradation. These effective rates have been measured using fluorescence recovery after photobleaching (FRAP) and photoactivation. To unravel which molecular events determine the effective rates, such tissue-level assays have been combined with genetic analysis, high-resolution assays, and models that take into account interactions with receptors, extracellular components and trafficking. Nevertheless, because of the natural and experimental data variability, and the underlying assumptions of transport models, it remains challenging to conclusively distinguish between cellular mechanisms. AU - Kicheva, Anna AU - Bollenbach, Mark Tobias AU - Wartlick, Ortrud AU - Julicher, Frank AU - Gonzalez Gaitan, Marcos ID - 2970 IS - 6 JF - Current Opinion in Genetics & Development TI - Investigating the principles of morphogen gradient formation: from tissues to cells VL - 22 ER -