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 - 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 - JOUR AB - Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior. AU - Chait, Remy P AU - Ruess, Jakob AU - Bergmiller, Tobias AU - Tkacik, Gasper AU - Guet, Calin C ID - 613 IS - 1 JF - Nature Communications SN - 20411723 TI - Shaping bacterial population behavior through computer interfaced control of individual cells VL - 8 ER - TY - JOUR AB - The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ~1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell. AU - Renault, Thibaud AU - Abraham, Anthony AU - Bergmiller, Tobias AU - Paradis, Guillaume AU - Rainville, Simon AU - Charpentier, Emmanuelle AU - Guet, Calin C AU - Tu, Yuhai AU - Namba, Keiichi AU - Keener, James AU - Minamino, Tohru AU - Erhardt, Marc ID - 655 JF - eLife SN - 2050084X TI - Bacterial flagella grow through an injection diffusion mechanism VL - 6 ER - TY - JOUR AB - While we have good understanding of bacterial metabolism at the population level, we know little about the metabolic behavior of individual cells: do single cells in clonal populations sometimes specialize on different metabolic pathways? Such metabolic specialization could be driven by stochastic gene expression and could provide individual cells with growth benefits of specialization. We measured the degree of phenotypic specialization in two parallel metabolic pathways, the assimilation of glucose and arabinose. We grew Escherichia coli in chemostats, and used isotope-labeled sugars in combination with nanometer-scale secondary ion mass spectrometry and mathematical modeling to quantify sugar assimilation at the single-cell level. We found large variation in metabolic activities between single cells, both in absolute assimilation and in the degree to which individual cells specialize in the assimilation of different sugars. Analysis of transcriptional reporters indicated that this variation was at least partially based on cell-to-cell variation in gene expression. Metabolic differences between cells in clonal populations could potentially reduce metabolic incompatibilities between different pathways, and increase the rate at which parallel reactions can be performed. AU - Nikolic, Nela AU - Schreiber, Frank AU - Dal Co, Alma AU - Kiviet, Daniel AU - Bergmiller, Tobias AU - Littmann, Sten AU - Kuypers, Marcel AU - Ackermann, Martin ID - 541 IS - 12 JF - PLoS Genetics SN - 15537390 TI - Cell-to-cell variation and specialization in sugar metabolism in clonal bacterial populations VL - 13 ER - TY - GEN AB - Estimates of 13 C-arabinose and 2 H-glucose uptake from the fractions of heavy isotopes measured in single cells AU - Nikolic, Nela AU - Schreiber, Frank AU - Dal Co, Alma AU - Kiviet, Daniel AU - Bergmiller, Tobias AU - Littmann, Sten AU - Kuypers, Marcel AU - Ackermann, Martin ID - 9845 TI - Mathematical model ER - TY - GEN AU - Nikolic, Nela AU - Schreiber, Frank AU - Dal Co, Alma AU - Kiviet, Daniel AU - Bergmiller, Tobias AU - Littmann, Sten AU - Kuypers, Marcel AU - Ackermann, Martin ID - 9846 TI - Supplementary methods ER - TY - GEN AU - Nikolic, Nela AU - Schreiber, Frank AU - Dal Co, Alma AU - Kiviet, Daniel AU - Bergmiller, Tobias AU - Littmann, Sten AU - Kuypers, Marcel AU - Ackermann, Martin ID - 9844 TI - Source data for figures and tables ER - TY - DATA AB - This repository contains the data collected for the manuscript "Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity". The data is compressed into a single archive. Within the archive, different folders correspond to figures of the main text and the SI of the related publication. Data is saved as plain text, with each folder containing a separate readme file describing the format. Typically, the data is from fluorescence microscopy measurements of single cells growing in a microfluidic "mother machine" device, and consists of relevant values (primarily arbitrary unit or normalized fluorescence measurements, and division times / growth rates) after raw microscopy images have been processed, segmented, and their features extracted, as described in the methods section of the related publication. AU - Bergmiller, Tobias AU - Andersson, Anna M AU - Tomasek, Kathrin AU - Balleza, Enrique AU - Kiviet, Daniel AU - Hauschild, Robert AU - Tkacik, Gasper AU - Guet, Calin C ID - 5560 KW - single cell microscopy KW - mother machine microfluidic device KW - AcrAB-TolC pump KW - multi-drug efflux KW - Escherichia coli TI - Biased partitioning of the multi-drug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity ER - TY - JOUR AB - The molecular mechanisms underlying phenotypic variation in isogenic bacterial populations remain poorly understood.We report that AcrAB-TolC, the main multidrug efflux pump of Escherichia coli, exhibits a strong partitioning bias for old cell poles by a segregation mechanism that is mediated by ternary AcrAB-TolC complex formation. Mother cells inheriting old poles are phenotypically distinct and display increased drug efflux activity relative to daughters. Consequently, we find systematic and long-lived growth differences between mother and daughter cells in the presence of subinhibitory drug concentrations. A simple model for biased partitioning predicts a population structure of long-lived and highly heterogeneous phenotypes. This straightforward mechanism of generating sustained growth rate differences at subinhibitory antibiotic concentrations has implications for understanding the emergence of multidrug resistance in bacteria. AU - Bergmiller, Tobias AU - Andersson, Anna M AU - Tomasek, Kathrin AU - Balleza, Enrique AU - Kiviet, Daniel AU - Hauschild, Robert AU - Tkacik, Gasper AU - Guet, Calin C ID - 665 IS - 6335 JF - Science SN - 00368075 TI - Biased partitioning of the multidrug efflux pump AcrAB TolC underlies long lived phenotypic heterogeneity VL - 356 ER - TY - JOUR AB - In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported. AU - Boehm, Alex AU - Arnoldini, Markus AU - Bergmiller, Tobias AU - Röösli, Thomas AU - Bigosch, Colette AU - Ackermann, Martin ID - 1250 IS - 4 JF - PLoS Genetics TI - Genetic manipulation of glycogen allocation affects replicative lifespan in E coli VL - 12 ER - TY - GEN AU - Boehm, Alex AU - Arnoldini, Markus AU - Bergmiller, Tobias AU - Röösli, Thomas AU - Bigosch, Colette AU - Ackermann, Martin ID - 9873 TI - Quantification of the growth rate reduction as a consequence of age-specific mortality ER - TY - JOUR AB - Parasitism creates selection for resistance mechanisms in host populations and is hypothesized to promote increased host evolvability. However, the influence of these traits on host evolution when parasites are no longer present is unclear. We used experimental evolution and whole-genome sequencing of Escherichia coli to determine the effects of past and present exposure to parasitic viruses (phages) on the spread of mutator alleles, resistance, and bacterial competitive fitness. We found that mutator alleles spread rapidly during adaptation to any of four different phage species, and this pattern was even more pronounced with multiple phages present simultaneously. However, hypermutability did not detectably accelerate adaptation in the absence of phages and recovery of fitness costs associated with resistance. Several lineages evolved phage resistance through elevated mucoidy, and during subsequent evolution in phage-free conditions they rapidly reverted to nonmucoid, phage-susceptible phenotypes. Genome sequencing revealed that this phenotypic reversion was achieved by additional genetic changes rather than by genotypic reversion of the initial resistance mutations. Insertion sequence (IS) elements played a key role in both the acquisition of resistance and adaptation in the absence of parasites; unlike single nucleotide polymorphisms, IS insertions were not more frequent in mutator lineages. Our results provide a genetic explanation for rapid reversion of mucoidy, a phenotype observed in other bacterial species including human pathogens. Moreover, this demonstrates that the types of genetic change underlying adaptation to fitness costs, and consequently the impact of evolvability mechanisms such as increased point-mutation rates, depend critically on the mechanism of resistance. AU - Wielgoss, Sébastien AU - Bergmiller, Tobias AU - Bischofberger, Anna M. AU - Hall, Alex R. ID - 5749 IS - 3 JF - Molecular Biology and Evolution SN - 0737-4038 TI - Adaptation to parasites and costs of parasite resistance in mutator and nonmutator bacteria VL - 33 ER - TY - JOUR AB - Restriction-modification (RM) systems represent a minimal and ubiquitous biological system of self/non-self discrimination in prokaryotes [1], which protects hosts from exogenous DNA [2]. The mechanism is based on the balance between methyltransferase (M) and cognate restriction endonuclease (R). M tags endogenous DNA as self by methylating short specific DNA sequences called restriction sites, whereas R recognizes unmethylated restriction sites as non-self and introduces a double-stranded DNA break [3]. Restriction sites are significantly underrepresented in prokaryotic genomes [4-7], suggesting that the discrimination mechanism is imperfect and occasionally leads to autoimmunity due to self-DNA cleavage (self-restriction) [8]. Furthermore, RM systems can promote DNA recombination [9] and contribute to genetic variation in microbial populations, thus facilitating adaptive evolution [10]. However, cleavage of self-DNA by RM systems as elements shaping prokaryotic genomes has not been directly detected, and its cause, frequency, and outcome are unknown. We quantify self-restriction caused by two RM systems of Escherichia coli and find that, in agreement with levels of restriction site avoidance, EcoRI, but not EcoRV, cleaves self-DNA at a measurable rate. Self-restriction is a stochastic process, which temporarily induces the SOS response, and is followed by DNA repair, maintaining cell viability. We find that RM systems with higher restriction efficiency against bacteriophage infections exhibit a higher rate of self-restriction, and that this rate can be further increased by stochastic imbalance between R and M. Our results identify molecular noise in RM systems as a factor shaping prokaryotic genomes. AU - Pleska, Maros AU - Qian, Long AU - Okura, Reiko AU - Bergmiller, Tobias AU - Wakamoto, Yuichi AU - Kussell, Edo AU - Guet, Calin C ID - 1243 IS - 3 JF - Current Biology TI - Bacterial autoimmunity due to a restriction-modification system VL - 26 ER - TY - GEN AB - Parasitism creates selection for resistance mechanisms in host populations and is hypothesized to promote increased host evolvability. However, the influence of these traits on host evolution when parasites are no longer present is unclear. We used experimental evolution and whole-genome sequencing of Escherichia coli to determine the effects of past and present exposure to parasitic viruses (phages) on the spread of mutator alleles, resistance, and bacterial competitive fitness. We found that mutator alleles spread rapidly during adaptation to any of four different phage species, and this pattern was even more pronounced with multiple phages present simultaneously. However, hypermutability did not detectably accelerate adaptation in the absence of phages and recovery of fitness costs associated with resistance. Several lineages evolved phage resistance through elevated mucoidy, and during subsequent evolution in phage-free conditions they rapidly reverted to nonmucoid, phage-susceptible phenotypes. Genome sequencing revealed that this phenotypic reversion was achieved by additional genetic changes rather than by genotypic reversion of the initial resistance mutations. Insertion sequence (IS) elements played a key role in both the acquisition of resistance and adaptation in the absence of parasites; unlike single nucleotide polymorphisms, IS insertions were not more frequent in mutator lineages. Our results provide a genetic explanation for rapid reversion of mucoidy, a phenotype observed in other bacterial species including human pathogens. Moreover, this demonstrates that the types of genetic change underlying adaptation to fitness costs, and consequently the impact of evolvability mechanisms such as increased point-mutation rates, depend critically on the mechanism of resistance. AU - Wielgoss, Sébastien AU - Bergmiller, Tobias AU - Bischofberger, Anna M. AU - Hall, Alex R. ID - 9719 TI - Data from: Adaptation to parasites and costs of parasite resistance in mutator and non-mutator bacteria ER - TY - JOUR AB - Gene duplication is important in evolution, because it provides new raw material for evolutionary adaptations. Several existing hypotheses about the causes of duplicate retention and diversification differ in their emphasis on gene dosage, subfunctionalization, and neofunctionalization. Little experimental data exist on the relative importance of gene expression changes and changes in coding regions for the evolution of duplicate genes. Furthermore, we do not know how strongly the environment could affect this importance. To address these questions, we performed evolution experiments with the TEM-1 beta lactamase gene in Escherichia coli to study the initial stages of duplicate gene evolution in the laboratory. We mimicked tandem duplication by inserting two copies of the TEM-1 gene on the same plasmid. We then subjected these copies to repeated cycles of mutagenesis and selection in various environments that contained antibiotics in different combinations and concentrations. Our experiments showed that gene dosage is the most important factor in the initial stages of duplicate gene evolution, and overshadows the importance of point mutations in the coding region. AU - Dhar, Riddhiman AU - Bergmiller, Tobias AU - Wagner, Andreas ID - 9931 IS - 6 JF - Evolution SN - 0014-3820 TI - Increased gene dosage plays a predominant role in the initial stages of evolution of duplicate TEM-1 beta lactamase genes VL - 68 ER - TY - GEN AB - Gene duplication is important in evolution, because it provides new raw material for evolutionary adaptations. Several existing hypotheses about the causes of duplicate retention and diversification differ in their emphasis on gene dosage, sub-functionalization, and neo-functionalization. Little experimental data exists on the relative importance of gene expression changes and changes in coding regions for the evolution of duplicate genes. Furthermore, we do not know how strongly the environment could affect this importance. To address these questions, we performed evolution experiments with the TEM-1 beta lactamase gene in E. coli to study the initial stages of duplicate gene evolution in the laboratory. We mimicked tandem duplication by inserting two copies of the TEM-1 gene on the same plasmid. We then subjected these copies to repeated cycles of mutagenesis and selection in various environments that contained antibiotics in different combinations and concentrations. Our experiments showed that gene dosage is the most important factor in the initial stages of duplicate gene evolution, and overshadows the importance of point mutations in the coding region. AU - Dhar, Riddhiman AU - Bergmiller, Tobias AU - Wagner, Andreas ID - 9932 TI - Data from: Increased gene dosage plays a predominant role in the initial stages of evolution of duplicate TEM-1 beta lactamase genes ER - TY - JOUR AB - High relatedness among interacting individuals has generally been considered a precondition for the evolution of altruism. However, kin-selection theory also predicts the evolution of altruism when relatedness is low, as long as the cost of the altruistic act is minor compared with its benefit. Here, we demonstrate evidence for a low-cost altruistic act in bacteria. We investigated Escherichia coli responding to the attack of an obligately lytic phage by committing suicide in order to prevent parasite transmission to nearby relatives. We found that bacterial suicide provides large benefits to survivors at marginal costs to committers. The cost of suicide was low, because infected cells are moribund, rapidly dying upon phage infection, such that no more opportunity for reproduction remains. As a consequence of its marginal cost, host suicide was selectively favoured even when relatedness between committers and survivors approached zero. Altogether, our findings demonstrate that low-cost suicide can evolve with ease, represents an effective host-defence strategy, and seems to be widespread among microbes. Moreover, low-cost suicide might also occur in higher organisms as exemplified by infected social insect workers leaving the colony to die in isolation. AU - Refardt, Dominik AU - Bergmiller, Tobias AU - Kümmerli, Rolf ID - 2853 IS - 1759 JF - Proceedings of the Royal Society of London Series B Biological Sciences TI - Altruism can evolve when relatedness is low: Evidence from bacteria committing suicide upon phage infection VL - 280 ER -