RESUMEN
The experimental challenges posed by integral membrane proteins hinder molecular understanding of transmembrane signaling mechanisms. Here, we exploited protein crosslinking assays in living cells to follow conformational and dynamic stimulus signals in Tsr, the Escherichia coli serine chemoreceptor. Tsr mediates serine chemotaxis by integrating transmembrane serine-binding inputs with adaptational modifications of a methylation helix bundle to regulate a signaling kinase at the cytoplasmic tip of the receptor molecule. We created a series of cysteine replacements at Tsr residues adjacent to hydrophobic packing faces of the bundle helices and crosslinked them with a cell-permeable, bifunctional thiol-reagent. We identified an extensively crosslinked dynamic junction midway through the methylation helix bundle that seemed uniquely poised to respond to serine signals. We explored its role in mediating signaling shifts between different packing arrangements of the bundle helices by measuring crosslinking in receptor molecules with apposed pairs of cysteine reporters in each subunit and assessing their signaling behaviors with an in vivo kinase assay. In the absence of serine, the bundle helices evinced compact kinase-ON packing arrangements; in the presence of serine, the dynamic junction destabilized adjacent bundle segments and shifted the bundle to an expanded, less stable kinase-OFF helix-packing arrangement. An AlphaFold 3 model of kinase-active Tsr showed a prominent bulge and kink at the dynamic junction that might antagonize stable structure at the receptor tip. Serine stimuli probably inhibit kinase activity by shifting the bundle to a less stably-packed conformation that relaxes structural strain at the receptor tip, thereby freezing kinase activity.
RESUMEN
In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.
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Quimiotaxis , Escherichia coli , Transducción de Señal , Escherichia coli/metabolismo , Escherichia coli/fisiología , Quimiotaxis/fisiología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Factores Quimiotácticos/metabolismoRESUMEN
Chemotactic bacteria not only navigate chemical gradients, but also shape their environments by consuming and secreting attractants. Investigating how these processes influence the dynamics of bacterial populations has been challenging because of a lack of experimental methods for measuring spatial profiles of chemoattractants in real time. Here, we use a fluorescent sensor for aspartate to directly measure bacterially generated chemoattractant gradients during collective migration. Our measurements show that the standard Patlak-Keller-Segel model for collective chemotactic bacterial migration breaks down at high cell densities. To address this, we propose modifications to the model that consider the impact of cell density on bacterial chemotaxis and attractant consumption. With these changes, the model explains our experimental data across all cell densities, offering insight into chemotactic dynamics. Our findings highlight the significance of considering cell density effects on bacterial behavior, and the potential for fluorescent metabolite sensors to shed light on the complex emergent dynamics of bacterial communities.
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Factores Quimiotácticos , Quimiotaxis , Transporte Biológico , Ácido Aspártico , ColorantesRESUMEN
The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.
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Quimiotaxis , Proteínas de Escherichia coli , Quimiotaxis/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligandos , Histidina Quinasa/metabolismo , Escherichia coli/metabolismo , Transducción de Señal/fisiología , Proteínas Bacterianas/metabolismoRESUMEN
Numerous antibiotic resistance genes (ARGs) and virulence factors (VFs) found in animal manure pose significant risks to human health. However, the effects of graphene sodium selenite (GSSe), a novel chemical nano-Selenium, and biological nano-Selenium (BNSSe), a new bioaugmentation nano-Se, on bacterial Se metabolism, chemotaxis, ARGs, and VFs in animal manure remain unknown. In this study, we investigated the effects of GSSe and BNSSe on ARGs and VFs expression in broiler manure using high-throughput sequencing. Results showed that BNSSe reduced Se pressure during anaerobic fermentation by inhibiting bacterial selenocompound metabolism pathways, thereby lowering manure Selenium pollution. Additionally, the expression levels of ARGs and VFs were lower in the BNSSe group compared to the Sodium Selenite and GSSe groups, as BNSSe inhibited bacterial chemotaxis pathways. Co-occurrence network analysis identified ARGs and VFs within the following phyla Bacteroidetes (genera Butyricimonas, Odoribacter, Paraprevotella, and Rikenella), Firmicutes (genera Lactobacillus, Candidatus_Borkfalkia, Merdimonas, Oscillibacter, Intestinimonas, and Megamonas), and Proteobacteria (genera Desulfovibrio). The expression and abundance of ARGs and VFs genes were found to be associated with ARGs-VFs coexistence. Moreover, BNSSe disruption of bacterial selenocompound metabolism and chemotaxis pathways resulted in less frequent transfer of ARGs and VFs. These findings indicate that BNSSe can reduce ARGs and VFs expression in animal manure by suppressing bacterial selenocompound metabolism and chemotaxis pathways.
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Selenio , Humanos , Animales , Selenio/farmacología , Estiércol/análisis , Genes Bacterianos , Antibacterianos/farmacología , Quimiotaxis/genética , Selenito de Sodio/farmacología , Pollos/genética , Bacterias , Farmacorresistencia Microbiana/genética , Bacteroidetes , FirmicutesRESUMEN
Animal manure contains many antibiotic resistance genes (ARGs) and virulence factors (VFs), posing significant health threats to humans. However, the effects of graphene nano zinc oxide (GZnONP), a zinc bioaugmentation substitute, on bacterial chemotaxis, ARGs, and VFs in animal manure remain scanty. Herein, the effect of GZnONP on the in vivo anaerobic expression of ARGs and VFs in cattle manure was assessed using high-throughput sequencing. Results showed that GZnONP inhibited bacterial chemotaxis by reducing the zinc pressure under anaerobic fermentation, altering the microbial community structure. The expression of ARGs was significantly lower in GZnONP than in zinc oxide and nano zinc oxide (ZnONP) groups. The expression of VFs was lower in the GZnONP than in the zinc oxide and ZnONP groups by 9.85 % and 13.46 %, respectively. Co-occurrence network analysis revealed that ARGs and VFs were expressed by the Spirochaetes phylum, Paraprevotella genus, and Treponema genus et al. The ARGs-VFs coexistence was related to the expression/abundance of ARGs and VFs genes. GZnONP reduces the abundance of certain bacterial species by disrupting chemotaxis, minimizing the transfer of ARGs and VFs. These findings suggest that GZnONP, a bacterial chemotaxis suppressor, effectively reduces the expression and release of ARGs and VFs in animal manure.
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Grafito , Óxido de Zinc , Humanos , Animales , Bovinos , Antibacterianos/farmacología , Zinc/farmacología , Estiércol/microbiología , Genes Bacterianos , Óxido de Zinc/farmacología , Farmacorresistencia Microbiana/genética , Bacterias/genéticaRESUMEN
Mitochondrial complex II is traditionally studied for its participation in two key respiratory processes: the electron transport chain and the Krebs cycle. There is now a rich body of literature explaining how complex II contributes to respiration. However, more recent research shows that not all of the pathologies associated with altered complex II activity clearly correlate with this respiratory role. Complex II activity has now been shown to be necessary for a range of biological processes peripherally related to respiration, including metabolic control, inflammation, and cell fate. Integration of findings from multiple types of studies suggests that complex II both participates in respiration and controls multiple succinate-dependent signal transduction pathways. Thus, the emerging view is that the true biological function of complex II is well beyond respiration. This review uses a semichronological approach to highlight major paradigm shifts that occurred over time. Special emphasis is given to the more recently identified functions of complex II and its subunits because these findings have infused new directions into an established field.
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Complejo II de Transporte de Electrones , Succinato Deshidrogenasa , Ciclo del Ácido Cítrico , Respiración , Transducción de Señal , Succinato Deshidrogenasa/metabolismo , Mitocondrias , Complejo II de Transporte de Electrones/metabolismoRESUMEN
The ability to control locomotion in a dynamic environment provides a competitive advantage for microorganisms, thus driving the evolution of sophisticated regulatory systems. In total, 19 known categories of chemotaxis systems control motility mediated by flagella or Type IV pili, plus other cellular functions. A key feature that distinguishes chemotaxis systems from generic two-component regulatory systems is separation of receptor and kinase functions into distinct proteins, linked by CheW scaffold proteins. This arrangement allows for formation of varied arrays with remarkable signaling properties. We recently analyzed sequences of CheW-like domains found in CheA kinases and CheW and CheV scaffold proteins. In total, 16 Architectures of CheA, CheW, and CheV proteins contain ~94% of all CheW-like domains and form six Classes with likely functional specializations. We surveyed chemotaxis system categories and proteins containing CheW-like domains in ~1900 prokaryotic species, the most comprehensive analysis to date, revealing new insights. Co-occurrence analyses suggested that many chemotaxis systems occur in non-random combinations within species, implying synergy or antagonism. Furthermore, many Architectures of proteins containing CheW-like domains occurred predominantly with specific categories of chemotaxis systems, suggesting specialized functional interactions. We propose Class 1 (~80%) and Class 6 (~20%) CheW proteins exhibit preferences for distinct chemoreceptor structures. Furthermore, rare (~1%) Class 2 CheW proteins frequently co-occurred with methyl-accepting coiled coil proteins, which contain both receptor and kinase functions and so do not require connection via a CheW scaffold but may benefit from arrays. Last, rare multidomain CheW proteins may interact with different receptors than single-domain CheW proteins.
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Quimiotaxis , Proteínas de Escherichia coli , Quimiotaxis/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Bacterianas/química , Transducción de Señal , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/química , Histidina Quinasa/genética , Histidina Quinasa/metabolismoRESUMEN
To swim and navigate, motile bacteria synthesize a complex motility machinery involving flagella, motors, and a sensory system. A myriad of studies has elucidated the molecular processes involved, but less is known about the coordination of motility expression with cellular physiology: In Escherichia coli, motility genes are strongly up-regulated in nutrient-poor conditions compared to nutrient-replete conditions; yet a quantitative link to cellular motility has not been developed. Here, we systematically investigated gene expression, swimming behavior, cell growth, and available proteomics data across a broad spectrum of exponential growth conditions. Our results suggest that cells up-regulate the expression of motility genes at slow growth to compensate for reduction in cell size, such that the number of flagella per cell is maintained across conditions. The observed four or five flagella per cell is the minimum number needed to keep the majority of cells motile. This simple regulatory objective allows E. coli cells to remain motile across a broad range of growth conditions, while keeping the biosynthetic and energetic demands to establish and drive the motility machinery at the minimum needed. Given the strong reduction in flagella synthesis resulting from cell size increases at fast growth, our findings also provide a different physiological perspective on bacterial cell size control: A larger cell size at fast growth is an efficient strategy to increase the allocation of cellular resources to the synthesis of those proteins required for biomass synthesis and growth, while maintaining processes such as motility that are only needed on a per-cell basis.
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Quimiotaxis , Proteínas de Escherichia coli , Escherichia coli , Quimiotaxis/genética , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión GénicaRESUMEN
Bacterial chemotaxis in unicellular Escherichia coli, the simplest biological creature, enables it to perform effective searching behaviour even with a single sensor, achieved via a sequence of "tumbling" and "swimming" behaviours guided by gradient information. Recent studies show that suitable random walk strategies may guide the behaviour in the absence of gradient information. This article presents a novel and minimalistic biologically inspired search strategy inspired by bacterial chemotaxis and embodied intelligence concept: a concept stating that intelligent behaviour is a result of the interaction among the "brain," body morphology including the sensory sensitivity tuned by the morphology, and the environment. Specifically, we present bacterial chemotaxis inspired searching behaviour with and without gradient information based on biological fluctuation framework: a mathematical framework that explains how biological creatures utilize noises in their behaviour. Via extensive simulation of a single sensor mobile robot that searches for a moving target, we will demonstrate how the effectiveness of the search depends on the sensory sensitivity and the inherent random walk strategies produced by the brain of the robot, comprising Ballistic, Levy, Brownian, and Stationary search. The result demonstrates the importance of embodied intelligence even in a behaviour inspired by the simplest creature.
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Escherichia coli , Inteligencia , Simulación por Computador , Modelos BiológicosRESUMEN
The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed in vivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the in vivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimidoethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during in vivo Förster resonance energy transfer-based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that in vivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule.
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Escherichia coli , Proteínas Quimiotácticas Aceptoras de Metilo/química , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Modelos Moleculares , Serina/metabolismoRESUMEN
Collective behaviors require coordination among a group of individuals. As a result, individuals that are too phenotypically different from the rest of the group can be left out, reducing heterogeneity, but increasing coordination. If individuals also reproduce, the offspring can have different phenotypes from their parent(s). This raises the question of how these two opposing processes-loss of diversity by collective behaviors and generation of it through growth and inheritance-dynamically shape the phenotypic composition of an isogenic population. We examine this question theoretically using collective migration of chemotactic bacteria as a model system, where cells of different swimming phenotypes are better suited to navigate in different environments. We find that the differential loss of phenotypes caused by collective migration is environment-dependent. With cell growth, this differential loss enables migrating populations to dynamically adapt their phenotype compositions to the environment, enhancing migration through multiple environments. Which phenotypes are produced upon cell division depends on the level of nongenetic inheritance, and higher inheritance leads to larger composition adaptation and faster migration at steady state. However, this comes at the cost of slower responses to new environments. Due to this trade-off, there is an optimal level of inheritance that maximizes migration speed through changing environments, which enables a diverse population to outperform a nondiverse one. Growing populations might generally leverage the selection-like effects provided by collective behaviors to dynamically shape their own phenotype compositions, without mutations.
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Bacterias , Evolución Biológica , Quimiotaxis , Adaptación Fisiológica/genética , FenotipoRESUMEN
Chemoreceptors are usually transmembrane proteins dedicated to the detection of compound gradients or signals in the surroundings of a bacterium. After detection, they modulate the activation of CheA-CheY, the core of the chemotactic pathway, to allow cells to move upwards or downwards depending on whether the signal is an attractant or a repellent, respectively. Environmental bacteria such as Shewanella oneidensis harbour dozens of chemoreceptors or MCPs (methyl-accepting chemotaxis proteins). A recent study revealed that MCP SO_1056 of S. oneidensis binds chromate. Here, we show that this MCP also detects an additional attractant (l-malate) and two repellents (nickel and cobalt). The experiments were performed in vivo by the agarose-in-plug technique after overproducing MCP SO_1056 and in vitro, when possible, by submitting the purified ligand-binding domain (LBD) of SO_1056 to a thermal shift assay (TSA) coupled to isothermal titration calorimetry (ITC). ITC assays revealed a KD of 3.4 µm for l-malate and of 47.7 µm for nickel. We conclude that MCP SO_1056 binds attractants and repellents of unrelated composition. The LBD of SO_1056 belongs to the double Cache_1 family and is highly homologous to PctA, a chemoreceptor from Pseudomonas aeruginosa that detects several amino acids. Therefore, LBDs of the same family can bind diverse compounds, confirming that experimental approaches are required to define accurate LBD-binding molecules or signals.
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Quimiotaxis , Malatos , Níquel , Proteínas Bacterianas/metabolismo , Proteínas Quimiotácticas Aceptoras de MetiloRESUMEN
To swim up gradients of nutrients, E. coli senses nutrient concentrations within its periplasm. For small nutrient molecules, periplasmic concentrations typically match extracellular concentrations. However, this is not necessarily the case for saccharides, such as maltose, which are transported into the periplasm via a specific porin. Previous observations have shown that, under various conditions, E. coli limits maltoporin abundance so that, for extracellular micromolar concentrations of maltose, there are predicted to be only nanomolar concentrations of free maltose in the periplasm. Thus, in the micromolar regime, the total uptake of maltose from the external environment into the cytoplasm is limited not by the abundance of cytoplasmic transport proteins but by the abundance of maltoporins. Here, we present results from experiments and modeling suggesting that this porin-limited transport enables E. coli to sense micromolar gradients of maltose despite having a high-affinity ABC transport system that is saturated at these micromolar levels. We used microfluidic assays to study chemotaxis of E. coli in various gradients of maltose and methyl-aspartate and leveraged our experimental observations to develop a mechanistic transport-and-sensing chemotaxis model. Incorporating this model into agent-based simulations, we discover a trade-off between uptake and sensing: although high-affinity transport enables higher uptake rates at low nutrient concentrations, it severely limits the range of dynamic sensing. We thus propose that E. coli may limit periplasmic uptake to increase its chemotactic sensitivity, enabling it to use maltose as an environmental cue.
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Proteínas de Escherichia coli , Proteínas de Unión Periplasmáticas , Proteínas Bacterianas/metabolismo , Quimiotaxis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Maltosa/metabolismo , Proteínas de Unión a Maltosa/metabolismo , Proteínas de Unión Periplasmáticas/metabolismo , Porinas/metabolismoRESUMEN
Polymers are important components of the complex fluid environment for microorganisms. The mechanical effects on bacterial motile behavior due to the viscous or viscoelastic properties of polymers were extensively studied, whereas possible chemical effects on bacterial motility through bacterial chemoreception of the polymers were unclear. Here we studied the chemotactic response ofEscherichia colito polymeric solutions by combining the bead assay and FRET measurements. We found that the wild-typeE. colistrain exhibited an attractant response to widely used polymers such as Ficoll 400, polyethylene glycol (PEG) 20000 and polyvinyl pyrrolidone (PVP) 360000, and the response amplitude from chemoreception was much larger than that from the load-dependence of motor switching due to viscosity change. The chemotactic response depended on the type of receptors and the chain length of the polymers. Our findings here provided important new ingredients for further studies of bacterial motile behavior in complex fluids.
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Polímeros , Povidona , Polímeros/química , Polímeros/farmacología , Povidona/química , ViscosidadRESUMEN
c-di-GMP is a ubiquitous bacterial second messenger that plays a central regulatory role in diverse biological processes. c-di-GMP was known to regulate chemotaxis in multiple bacterial species, but its effect on Escherichia coli chemotaxis remained unclear. As an effector of c-di-GMP in E. coli, YcgR when bound with c-di-GMP interacts with the flagellar motor to reduce its speed and its probability of rotating clockwise (CW bias). Here, we found that a significant fraction of the c-di-GMP::YcgR dynamically exchange between the motor and the cytosol. Through fluorescent measurements, we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR to the motor. To test the influence of elevated c-di-GMP levels on the chemotaxis pathway, we measured the chemotactic responses of E. coli cells using a FRET assay, finding that elevated c-di-GMP levels had no effect on the upstream part of chemotaxis pathway down to the level of CheY-P concentration. This suggested that the possible effect of elevated c-di-GMP levels on chemotactic motion was through regulation of motor speed and CW bias. Using stochastic simulations of chemotactic swimming, we showed that the effects of reducing motor speed and decreasing CW bias on chemotactic drift velocity are compensating for each other, resulting in minimal effect of elevated c-di-GMP levels on E. coli chemotaxis. Therefore, elevated c-di-GMP levels promote the transition from motile to sedentary forms of bacterial life by reducing the bacterial swimming speed and CW bias, while still maintaining a nearly intact chemotaxis capability in E. coli. IMPORTANCE The ubiquitous bacterial second messenger c-di-GMP was known to regulate chemotaxis in many bacterial species, but its effect on E. coli chemotaxis was unclear. Here we studied the effect of elevated c-di-GMP levels on chemotaxis in E. coli. We found that the binding of c-di-GMP::YcgR (its effector) and the chemotaxis response regulator CheY-P to the flagellar motor are noncompetitive, and elevated c-di-GMP levels do not affect the upstream part of the chemotaxis pathway down to the level of CheY-P concentration. Elevated c-di-GMP levels exert direct effects on the flagellar motor by reducing its speed and CW bias, but the resulting effects on chemotaxis performance are compensating for each other. Our findings here showed that elevated c-di-GMP levels maintain a nearly intact chemotaxis capability when promoting the transition from motile to sedentary forms of bacterial life in E. coli.
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Proteínas de Escherichia coli , Escherichia coli , Proteínas Bacterianas/química , Quimiotaxis/fisiología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Flagelos/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
SignificanceHost-emitted stress hormones significantly influence the growth and behavior of various bacterial species; however, their cellular targets have so far remained elusive. Here, we used customized probes and quantitative proteomics to identify the target of epinephrine and the α-adrenoceptor agonist phenylephrine in live cells of the aquatic pathogen Vibrio campbellii. Consequently, we have discovered the coupling protein CheW, which is in the center of the chemotaxis signaling network, as a target of both molecules. We not only demonstrate direct ligand binding to CheW but also elucidate how this affects chemotactic control. These findings are pivotal for further research on hormone-specific effects on bacterial behavior.
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Proteínas Bacterianas/metabolismo , Catecolaminas/fisiología , Factores Quimiotácticos/fisiología , Quimiotaxis/fisiología , Vibrio/fisiología , Catecoles/química , Factores Quimiotácticos/metabolismo , Hierro/análisis , Sondas Moleculares/química , Unión Proteica , Proteómica/métodos , Transducción de SeñalRESUMEN
Periplasmic binding proteins such as ribose-binding proteins (RBPs) are involved in the bacterial chemotaxis two-component system. RBP selectively identifies and interacts with ribose to induce a conformational change that leads to chemotaxis. Here, we report the development of an engineered Escherichia coli (E. coli) strain expressing a redesigned RBP that can effectively sense cadmium ions and regulate chemotactic movement of cells toward a cadmium ion gradient. RBP was computationally redesigned to bind cadmium ions and produce the conformational change required for chemoreceptor binding. The successful design, CdRBP1, binds to cadmium ions with a dissociation constant of 268 nM. When CdRBP1 was expressed in the periplasmic space of E. coli, the bacteria became live cadmium ion hunters with high selectivity over other divalent metal ions. This work presents an example of making cadmium ions, which are toxic for most organisms, as an attractant to regulate cells movement. Our approach also demonstrates that RBP can be precisely designed to develop metal-detecting living systems for potential applications in synthetic biology and environmental studies. IMPORTANCE Cadmium pollution is one of the major environmental problems due to excessive release and accumulation. New technologies that can auto-detect cadmium ions with good biocompatibility are in urgent need. In this study, we engineered the bacterial chemotaxis system to positively sense cadmium ions by redesigning ribose-binding protein (RBP) to tightly bind cadmium ion and produce the right conformational change for receptor binding and signaling. Our engineered E. coli cells can auto-detect and chase cadmium ions with divalent metal ion selectivity. Many attempts have been carried out to redesign RBP at the ribose binding site with little success. Instead of the ribose binding site, we introduced the cadmium binding site in the opening of the ribose binding pocket by a specially developed computational algorithm. Our design strategy can be applied to engineer live bacteria with autonomous detection and remediation abilities for metal ions or other chemicals in the future.
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Proteínas Portadoras , Proteínas de Escherichia coli , Proteínas Portadoras/química , Escherichia coli , Cadmio/metabolismo , Ribosa/metabolismo , Proteínas de Escherichia coli/química , Bacterias/metabolismoRESUMEN
Naphthenic acids (NAs) are a persistent toxic organic pollutant that occur in different environment worldwide and cause serious threat to the ecosystem and public health. However, knowledge on the behavior and fate of NAs in marine environments still remains unknown. In this study, the degradation mechanism of NAs (cyclohexylacetic acid, CHAA) was investigated using an common indigenous marine Pseudoalteromonas sp. The results showed that CHAA could be degraded completely under aerobic condition, but could not be utilized directly under anaerobic condition. Interestingly, transcriptome and key enzyme activity results showed the CHAA degradation pathway induced under aerobic condition could still work in anaerobic condition. The degradation was activated by acetyl-CoA transferase and sequentially formed the corresponding cyclohexene, alcohol, and ketone with the assistance of related enzymes, and finally cleaved by hydroxymethylglutarate-CoA lyase. Besides, there was a positive correlation between chemotaxis and aerobic degradation genes (r = 0.976, P < 0.05), the chemotaxis would enhance bacterium movement and NAs biodegradation. It is proposed that bacterium could translocate to NAs and accomplish biodegradation from aerobic to anaerobic environments, which was a new anaerobic degradation pathway of NAs. This study provides new insights into the fate of NAs and other organic contaminants in marine environment.
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Pseudoalteromonas , Biodegradación Ambiental , Ácidos Carboxílicos , Ecosistema , Pseudoalteromonas/genéticaRESUMEN
Coordination of diverse individuals often requires sophisticated communications and high-order computational abilities. Microbial populations can exhibit diverse individualistic behaviors, and yet can engage in collective migratory patterns with a spatially sorted arrangement of phenotypes. However, it is unclear how such spatially sorted patterns emerge from diverse individuals without complex computational abilities. Here, by investigating the single-cell trajectories during group migration, we discovered that, despite the constant migrating speed of a group, the drift velocities of individual bacteria decrease from the back to the front. With a Langevin-type modeling framework, we showed that this decreasing profile of drift velocities implies the spatial modulation of individual run-and-tumble random motions, and enables the bacterial population to migrate as a pushed wave front. Theoretical analysis and stochastic simulations further predicted that the pushed wave front can help a diverse population to stay in a tight group, while diverse individuals perform the same type of mean reverting processes around centers orderly aligned by their chemotactic abilities. This mechanism about the emergence of orderly collective migration from diverse individuals is experimentally demonstrated by titration of bacterial chemoreceptor abundance. These results reveal a simple computational principle for emergent ordered behaviors from heterogeneous individuals.
Organisms living in large groups often have to move together in order to navigate, forage for food, and increase their roaming range. Such groups are often made up of distinct individuals that must integrate their different behaviors in order to migrate in the same direction at a similar pace. For instance, for the bacteria Escherichia coli to travel as a condensed group, they must coordinate their response to a set of chemical signals called chemoattractants that tell them where to go. The chemoattractants surrounding the bacteria are unequally distributed so that there is more of them at the front than the back of the group. During migration, each bacterium moves towards this concentration gradient in a distinct way, spontaneously rotating its direction in a 'run-and-tumble' motion that guides it towards areas where there are high levels of these chemical signals. In addition to this variability, how well individual bacteria are able to swim up the gradient also differs within the population. Bacteria that are better at sensing the chemoattractant gradient are placed at the front of the group, while those that are worst are shifted towards the back. This spatial arrangement is thought to help the bacteria migrate together. But how E. coli organize themselves in to this pattern is unclear, especially as they cannot communicate directly with one another and display such diverse, randomized behaviors. To help answer this question, Bai, He et al. discovered a general principle that describes how single bacterial cells move within a group. The results showed that E. coli alter their run-and-tumble motion depending on where they reside within the population: individuals at the rear drift faster so they can catch up with the group, while those leading the group drift slower to draw themselves back. This 'reversion behavior' allows the migrating bacteria to travel at a constant speed around a mean position relative to the group. A cell's drifting speed is determined by how well it moves towards the chemoattractant and its response to the concentration gradient. As a result, the mean position around which the bacterium accelerates or deaccelerates will vary depending on how sensitive it is to the chemoattractant gradient. The E. coli therefore spatially arrange themselves so that the more sensitive bacteria are located at the front of the group where the gradient is shallower; and cells that are less sensitive are located towards the back where the gradient is steeper. These findings suggest a general principle for how bacteria form ordered patterns whilst migrating as a collective group. This behavior could also apply to other populations of distinct individuals, such as ants following a trail or flocks of birds migrating in between seasons.