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Microbial communities are ubiquitous in nature and play an important role in ecology and human health. Cross-feeding is thought to be core to microbial communities, though it remains unclear precisely why it emerges. Why have multi-species microbial communities evolved in many contexts and what protects microbial consortia from invasion? Here, we review recent insights into the emergence and stability of coexistence in microbial communities. A particular focus is the long-term evolutionary stability of coexistence, as observed for microbial communities that spontaneously evolved in the E. coli long-term evolution experiment (LTEE). We analyze these findings in the context of recent work on trade-offs between competing microbial objectives, which can constitute a mechanistic basis for the emergence of coexistence. Coexisting communities, rather than monocultures of the 'fittest' single strain, can form stable endpoints of evolutionary trajectories. Hence, the emergence of coexistence might be an obligatory outcome in the evolution of microbial communities. This implies that rather than embodying fragile metastable configurations, some microbial communities can constitute formidable ecosystems that are difficult to disrupt.
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In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained3, although recent works have revealed that these processes are indeed coupled4,5. Here, we report a striking increase of turgor pressure with growth rate in E. coli, suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics6.
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Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate, at least for certain 'poor' substrates. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. This result falls in line with previous observations of more subtle growth rate improvement for many other carbon sources. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.
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Escherichia coli , Manosa , Escherichia coli/genética , Manosa/metabolismo , Proteómica , Bacterias , EcosistemaRESUMEN
Membrane potential is a property of all living cells1. However, its physiological role in non-excitable cells is poorly understood. Resting membrane potential is typically considered fixed for a given cell type and under tight homeostatic control2, akin to body temperature in mammals. Contrary to this widely accepted paradigm, we found that membrane potential is a dynamic property that directly reflects tissue density and mechanical forces acting on the cell. Serving as a quasi-instantaneous, global readout of density and mechanical pressure, membrane potential is integrated with signal transduction networks by affecting the conformation and clustering of proteins in the membrane3,4, as well as the transmembrane flux of key signaling ions5,6. Indeed, we show that important mechano-sensing pathways, YAP, Jnk and p387-121314, are directly controlled by membrane potential. We further show that mechano-transduction via membrane potential plays a critical role in the homeostasis of epithelial tissues, setting tissue density by controlling proliferation and cell extrusion of cells. Moreover, a wave of depolarization triggered by mechanical stretch enhances the speed of wound healing. Mechano-transduction via membrane potential likely constitutes an ancient homeostatic mechanism in multi-cellular organisms, potentially serving as a steppingstone for the evolution of excitable tissues and neuronal mechano-sensing. The breakdown of membrane potential mediated homeostatic regulation may contribute to tumor growth.
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High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.
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Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento/métodos , Diagnóstico por Imagen , Impedancia Eléctrica , ElectrodosRESUMEN
In all growing cells, the cell envelope must expand in concert with cytoplasmic biomass to prevent lysis or molecular crowding. The complex cell wall of microbes and plants makes this challenge especially daunting and it unclear how cells achieve this coordination. Here, we uncover a striking linear increase of cytoplasmic pressure with growth rate in E. coli. Remarkably, despite this increase in turgor pressure with growth rate, cellular biomass density was constant across a wide range of growth rates. In contrast, perturbing pressure away from this scaling directly affected biomass density. A mathematical model, in which endopeptidase-mediated cell wall fluidization enables turgor pressure to set the pace of cellular volume expansion, not only explains these confounding observations, but makes several surprising quantitative predictions that we validated experimentally. The picture that emerges is that changes in turgor pressure across growth rates are mediated by counterions of ribosomal RNA. Profoundly, the coupling between rRNA and cytoplasmic pressure simultaneously coordinates cell wall expansion across growth rates and exerts homeostatic feedback control on biomass density. Because ribosome content universally scales with growth rate in fast growing cells, this universal mechanism may control cell wall biosynthesis in microbes and plants and drive the expansion of ribosome-addicted tumors that can exert substantial mechanical forces on their environment.
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According to a widely accepted paradigm of microbiology, steady-state growth rates are determined solely by current growth conditions1-3 and adaptations between growth states are rapid, as recently recapitulated by simple resource allocation models4. However, even in microbes overlapping regulatory networks can yield multi-stability or long-term cellular memory. Species like Listeria monocytogenes5 and Bacillus subtilis "distinguish" distinct histories for the commitment to sporulation6, but it is unclear if these states can persist over many generations. Remarkably, studying carbon co-utilization of Escherichia coli, we found that growth rates on combinations of carbon sources can depend critically on the previous growth condition. Growing in identical conditions, we observed differences in growth rates of up to 25% and we did not observe convergence of growth rates over 15 generations. We observed this phenomenon occurs across combinations of different phosphotransferase (PTS) substrates with various gluconeogenic carbon sources and found it to depend on the transcription factor Mlc.
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Bacteria like E. coli grow at vastly different rates on different substrates, however, the precise reason for this variability is poorly understood. Different growth rates have been attributed to 'nutrient quality', a key parameter in bacterial growth laws. However, it remains unclear to what extent nutrient quality is rooted in fundamental biochemical constraints like the energy content of nutrients, the protein cost required for their uptake and catabolism, or the capacity of the plasma membrane for nutrient transporters. Here, we show that while nutrient quality is indeed reflected in protein investment in substrate-specific transporters and enzymes, this is not a fundamental limitation on growth rate. We show that it is possible to turn mannose, one of the 'poorest' substrates of E. coli, into one of the 'best' substrates by reengineering chromosomal promoters of the mannose transporter and metabolic enzymes required for mannose degradation. However, we show that this faster growth rate comes at the cost of diverse cellular capabilities, reflected in longer lag phases, worse starvation survival and lower motility. We show that addition of cAMP to the medium can rescue these phenotypes but imposes a corresponding growth cost. Based on these data, we propose that nutrient quality is largely a self-determined, plastic property that can be modulated by the fraction of proteomic resources devoted to a specific substrate in the much larger proteome sector of catabolically activated genes. Rather than a fundamental biochemical limitation, nutrient quality reflects resource allocation decisions that are shaped by evolution in specific ecological niches and can be quickly adapted if necessary.
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Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.
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Evolution of complex communities of coexisting microbes remains poorly understood. The long-term evolution experiment on Escherichia coli (LTEE) revealed the spontaneous emergence of stable coexistence of multiple ecotypes, which persisted for more than 14,000 generations of continuous evolution. Here, using a combination of experiments and computer simulations, we show that the emergence and persistence of this phenomenon can be explained by the combination of two interacting trade-offs, rooted in biochemical constraints: First, faster growth is enabled by higher fermentation and obligate acetate excretion. Second, faster growth results in longer lag times when utilizing acetate after glucose is depleted. This combination creates an ecological niche for a slower-growing ecotype, specialized in switching to acetate. These findings demonstrate that trade-offs can give rise to surprisingly complex communities with evolutionarily stable coexistence of multiple variants in even the simplest environments.
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Ecosistema , Ecotipo , Escherichia coli/genética , Simulación por Computador , Acetatos , Evolución BiológicaRESUMEN
Bacteria reorganize their physiology upon entry to stationary phase. What part of this reorganization improves starvation survival is a difficult question because the change in physiology includes a global reorganization of the proteome, envelope, and metabolism of the cell. In this work, we used several trade-offs between fast growth and long survival to statistically score over 2,000 Escherichia coli proteins for their global correlation with death rate. The combined ranking allowed us to narrow down the set of proteins that positively correlate with survival and validate the causal role of a subset of proteins. Remarkably, we found that important survival genes are related to the cell envelope, i.e., periplasm and outer membrane, because the maintenance of envelope integrity of E. coli plays a crucial role during starvation. Our results uncover a new protective feature of the outer membrane that adds to the growing evidence that the outer membrane is not only a barrier that prevents abiotic substances from reaching the cytoplasm but also essential for bacterial proliferation and survival.
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Escherichia coli , Proteoma , Escherichia coli/genéticaRESUMEN
Adaptive stress resistance in microbes is mostly attributed to the expression of stress response genes, including heat-shock proteins. Here, we report a response of E. coli to heat stress caused by degradation of an enzyme in the methionine biosynthesis pathway (MetA). While MetA degradation can inhibit growth, which by itself is detrimental for fitness, we show that it directly benefits survival at temperatures exceeding 50°C, increasing survival chances by more than 1,000-fold. Using both experiments and mathematical modeling, we show quantitatively how protein expression, degradation rates, and environmental stressors cause long-term growth inhibition in otherwise habitable conditions. Because growth inhibition can be abolished with simple mutations, namely point mutations of MetA and protease knockouts, we interpret the breakdown of methionine synthesis as a system that has evolved to halt growth at high temperatures, analogous to "thermal fuses" in engineering that shut off electricity to prevent overheating.
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Proteínas de Escherichia coli , Escherichia coli , Respuesta al Choque Térmico , Homoserina O-Succiniltransferasa , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Calor , Metionina/metabolismo , TemperaturaRESUMEN
Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.
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Microscopía Óptica no Lineal , Espectrometría Raman , Metabolismo de los Lípidos , Lípidos , Microscopía/métodos , Proteínas , Espectrometría Raman/métodosRESUMEN
Electrode-based impedance and electrochemical measurements can provide cell-biology information that is difficult to obtain using optical-microscopy techniques. Such electrical methods are non-invasive, label-free, and continuous, eliminating the need for fluorescence reporters and overcoming optical imaging's throughput/temporal resolution limitations. Nonetheless, electrode-based techniques have not been heavily employed because devices typically contain few electrodes per well, resulting in noisy aggregate readouts. Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) have sometimes been used for electrophysiological measurements with thousands of electrodes per well at sub-cellular pitches, but only basic impedance mappings of cell attachment have been performed outside of electrophysiology. Here, we report on new field-based impedance mapping and electrochemical mapping/patterning techniques to expand CMOS-MEA cell-biology applications. The methods enable accurate measurement of cell attachment, growth/wound healing, cell-cell adhesion, metabolic state, and redox properties with single-cell spatial resolution (20 µm electrode pitch). These measurements allow the quantification of adhesion and metabolic differences of cells expressing oncogenes versus wild-type controls. The multi-parametric, cell-population statistics captured by the chip-scale integrated device opens up new avenues for fully electronic high-throughput live-cell assays for phenotypic screening and drug discovery applications.
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Técnicas de Cultivo de Célula , Semiconductores , Fenómenos Electrofisiológicos , Microelectrodos , ÓxidosRESUMEN
Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic reorganization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis, and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. We combined growth dynamics and dynamic changes in intracellular metabolite levels with a coarse-grained model that integrates fluxes, regulation, protein synthesis, and growth and uncovered fundamental limitations of the regulatory network: After nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade-offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.
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Gluconeogénesis , Pseudomonas putida , Carbono , Glucosa , Glucólisis , Pseudomonas putida/genéticaRESUMEN
This piece discusses how the different observations of two independent studies (Kotte et al, 2014; Basan et al, 2020), regarding population-level heterogeneity and lag times during diauxic shift, can be largely explained by different experimental protocols.
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Bacterias , Carbono , Adaptación Fisiológica , Bacterias/genética , Escherichia coliRESUMEN
The rate of cell growth is crucial for bacterial fitness and drives the allocation of bacterial resources, affecting, for example, the expression levels of proteins dedicated to metabolism and biosynthesis1,2. It is unclear, however, what ultimately determines growth rates in different environmental conditions. Moreover, increasing evidence suggests that other objectives are also important3-7, such as the rate of physiological adaptation to changing environments8,9. A common challenge for cells is that these objectives cannot be independently optimized, and maximizing one often reduces another. Many such trade-offs have indeed been hypothesized on the basis of qualitative correlative studies8-11. Here we report a trade-off between steady-state growth rate and physiological adaptability in Escherichia coli, observed when a growing culture is abruptly shifted from a preferred carbon source such as glucose to fermentation products such as acetate. These metabolic transitions, common for enteric bacteria, are often accompanied by multi-hour lags before growth resumes. Metabolomic analysis reveals that long lags result from the depletion of key metabolites that follows the sudden reversal in the central carbon flux owing to the imposed nutrient shifts. A model of sequential flux limitation not only explains the observed trade-off between growth and adaptability, but also allows quantitative predictions regarding the universal occurrence of such tradeoffs, based on the opposing enzyme requirements of glycolysis versus gluconeogenesis. We validate these predictions experimentally for many different nutrient shifts in E. coli, as well as for other respiro-fermentative microorganisms, including Bacillus subtilis and Saccharomyces cerevisiae.
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Adaptación Fisiológica , Ambiente , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Acetatos/metabolismo , Bacillus subtilis/citología , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , División Celular , Escherichia coli/enzimología , Escherichia coli/genética , Fermentación , Gluconeogénesis , Glucosa/metabolismo , Glucólisis , Metabolómica , Modelos Biológicos , Mutación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
Elucidating strategies of resource allocation and metabolism is crucial for a better understanding of microbial phenotypes. In particular, uncovering the governing principles underlying these processes would be a crucial step for achieving a central aim of systems microbiology, which is to quantitatively predict phenotypes of microbial cells or entire populations in diverse conditions. Here, some of the key concepts for understanding cellular resource allocation and metabolism that have been suggested over the past years are reviewed. In particular, recent experimental studies that have shown how phenotypic patterns from orthogonal genetic and environmental perturbations can help to differentiate between competing hypotheses and their respective predictions are discussed. Phenomenological models have proven to be a valuable addition to genome-scale models, capable of making quantitative predictions with only few parameters and having aided the identification of molecular mechanisms.
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Bacterias/metabolismo , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , FenotipoRESUMEN
Overflow metabolism in the presence of oxygen occurs at fast growth rates in a wide range of organisms including bacteria, yeast and cancer cells and plays an important role in biotechnology during production of proteins or metabolic compounds. As recently suggested, overflow metabolism can be understood in terms of proteome allocation, since fermentation has lower proteome cost for energy production than respiration. Here, we demonstrate that ArcA overexpression in aerobic conditions, results in downregulation of respiratory pathways and enhanced growth rates on glycolytic substrates of E. coli, coinciding with acetate excretion and increased carbon uptake rates. These results suggest that fermentation enables faster growth and demonstrate that fermentation on many glycolytic carbon sources is not limited by carbon uptake. Hence, these findings are difficult to reconcile with many alternative hypotheses that have been proposed for the origin of overflow metabolism and the growth rate dependence of fermentation and respiration, which are based on limited capacity of respiration or limitations in uptake rates and catabolic pathways. Instead, as suggested by increased lag phases of ArcA overexpression strains, respiratory energy metabolism may be related to a general preparatory response, observed for decreasing growth rates, but with limited advantages for maximizing steady-state growth rate.