RESUMO
Motile bacteria use large receptor arrays to detect chemical and physical stimuli in their environment, process this complex information, and accordingly bias their swimming in a direction they deem favorable. The chemoreceptor molecules form tripod-like trimers of receptor dimers through direct contacts between their cytoplasmic tips. A pair of trimers, together with a dedicated kinase enzyme, form a core signaling complex. Hundreds of core complexes network to form extended arrays. While considerable progress has been made in revealing the hierarchical structure of the array, the molecular properties underlying signal processing in these structures remain largely unclear. Here we analyzed the signaling properties of nonnetworked core complexes in live cells by following both conformational and kinase control responses to attractant stimuli and to output-biasing lesions at various locations in the receptor molecule. Contrary to the prevailing view that individual receptors are binary two-state devices, we demonstrate that conformational coupling between the ligand binding and the kinase-control receptor domains is, in fact, only moderate. In addition, we demonstrate communication between neighboring receptors through their trimer-contact domains that biases them to adopt similar signaling states. Taken together, these data suggest a view of signaling in receptor trimers that allows significant signal integration to occur within individual core complexes.
Assuntos
Proteínas de Escherichia coli , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Células Quimiorreceptoras/metabolismo , Proteínas de Transporte/metabolismo , Quimiotaxia/fisiologia , Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismoRESUMO
Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo These exciting experimental advances were just some of the many novel findings presented at the 15th Bacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019.
Assuntos
Bactérias/patogenicidade , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Bactérias/metabolismo , Aderência Bacteriana , Congressos como Assunto , Flagelos/fisiologia , Locomoção , Transdução de SinaisRESUMO
The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Mutagênese Sítio-DirigidaRESUMO
Bacterial chemoreceptors sense environmental stimuli and govern cell movement by transmitting the information to the flagellar motors. The highly conserved cytoplasmic domain of chemoreceptors consists in an alpha-helical hairpin that forms in the homodimer a coiled-coil four-helix bundle. Several classes of chemoreceptors that differ in the length of the coiled-coil structure were characterized. Many bacterial species code for chemoreceptors that belong to different classes, but how these receptors are organized and function in the same cell remains an open question. E. coli cells normally code for single class chemoreceptors that form extended arrays based on trimers of dimers interconnected by the coupling protein CheW and the kinase CheA. This structure promotes effective coupling between the different receptors in the modulation of the kinase activity. In this work, we engineered functional derivatives of the Tsr chemoreceptor of E. coli that mimic receptors whose cytoplasmic domain is longer by two heptads. We found that these long Tsr receptors did not efficiently mix with the native receptors and appeared to function independently. Our results suggest that the assembly of membrane-bound receptors of different specificities into mixed clusters is dictated by the length-class to which the receptors belong, ensuring cooperative function only between receptors of the same class.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Transdução de Sinais , Estresse Fisiológico , Proteínas de Bactérias/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Histidina Quinase , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de MetilRESUMO
The clustering of membrane-bound receptors plays an essential role in various biological systems. A notable model system for studying this phenomenon is the bacterial chemosensory cluster that allows motile bacteria to navigate along chemical gradients in their environment. While the basic structure of these chemosensory clusters is becoming clear, their dynamic nature and operation are not yet understood. By measuring the fluorescence polarization of tagged receptor clusters in live Escherichia coli cells, we provide evidence for stimulus-induced dynamics in these sensory clusters. We find that when a stimulus is applied, the packing of the receptors slowly decreases and that the process reverses when the stimulus is removed. Consistent with these physical changes we find that the effective cooperativity of the kinase response slowly evolves in the presence of a stimulus. Time-lapse fluorescence imaging indicates that, despite these changes, the receptor clusters do not generally dissociate upon ligand binding. These data reveal stimulus-dependent plasticity in chemoreceptor clusters.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Proteínas Quimiotáticas Aceptoras de Metil , Modelos Moleculares , Imagem Óptica , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
To cope in complex environments, motile bacteria have developed a chemosensory system that integrates multiple cues and directs their motion toward regions that it deems favorable. However, we have a limited understanding of the principles that govern bacterial behavior in complex stimuli fields. Here, we followed the spatial redistribution of E. coli cells in perplexing environments created by a local source of both beneficial (nutrients) and hazardous (low pH or indole) effectors. We identified two fundamentally distinct collective responses: a 'trade-off' response, in which bacteria sharply accumulated at a distance from the source that reflected a trade-off between the propagating effectors, and a 'bet-hedging' response, in which part of the bacteria accumulated away from the source, avoiding the hazardous effector, while the other part evaded the repulsive force and accumulated at the source. In addition, we demonstrate that cells lacking the Tsr sensor swim toward both repellents and, surprisingly, even toward pH values well below 7. Using a numerical analysis, we could correlate the collective bacterial responses with fundamentally distinct chemotactic force fields created along the channel by the propagation of the effectors and their unique perception by the chemosensory system.
Assuntos
Quimiotaxia , Escherichia coli , Bactérias , Quimiotaxia/fisiologia , Escherichia coli/fisiologiaRESUMO
Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple "two-state" model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.
Assuntos
Proteínas de Bactérias/química , Células Quimiorreceptoras/química , Quimiotaxia , Anisotropia , Proteínas de Bactérias/fisiologia , Dimerização , Escherichia coli K12/química , Escherichia coli K12/genética , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Modelos Químicos , Ligação Proteica , Receptores de Aminoácido/química , Receptores de Superfície Celular/fisiologia , Transdução de SinaisRESUMO
The anisotropy of the fluorescence emitted from fluorescent proteins, such as yellow fluorescent protein (YFP), is sensitive to Homo-FRET between the proteins. This effect can be used to detect in vivo ligand-induced changes in packing or conformation of tagged chemoreceptors. Such measurements of clustered or dispersed core-signaling units revealed quantitative dose-dependent responses of these sensors.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Fatores Quimiotáticos/química , Fatores Quimiotáticos/metabolismo , Proteínas Luminescentes/metabolismo , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/química , Quimiotaxia , Polarização de Fluorescência , Ligantes , Conformação Proteica , Transdução de SinaisRESUMO
The bacterial chemosensory arrays are a notable model for studying the basic principles of receptor clustering and cellular organization. Here, we provide a new perspective regarding the long-term dynamics of these clusters in growing E. coli cells. We demonstrate that pre-existing lateral clusters tend to avoid translocation to pole regions and, therefore, continually shuttle between the cell poles for many generations while being static relative to the local cell-wall matrix. We also show that the polar preference of clusters results fundamentally from reduced clustering efficiency in the lateral region, rather than a developmental-like progression of clusters. Furthermore, polar preference is surprisingly robust to structural alterations designed to probe preference due to curvature sorting, perturbing the cell envelope physiology affects the cluster-size distribution, and the size-dependent mobility of receptor complexes differs between polar and lateral regions. Thus, distinct envelope physiology in the polar and lateral cell regions may contribute to polar preference.
Assuntos
Células Quimiorreceptoras/metabolismo , Escherichia coli/metabolismo , Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
The two-component pathway in Escherichia coli chemotaxis has become a paradigm for bacterial signal processing. Genetics and biochemistry of the pathway as well as physiological responses have been studied in detail. Despite its relative simplicity, the chemotaxis pathway is renowned for its ability to amplify and integrate weak signals and for its robustness against various kinds of perturbations. All this information inspired multiple attempts at mathematical analysis and computer modeling, but a quantitative understanding of the pathway was hampered by our inability to follow the signal processing in vivo. To address this problem, we developed assays based on fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) that enabled us to monitor activity-dependent protein interactions in real time directly in living cells. Here, we describe quantitative applications of these assays in cell populations and on a single-cell level to study the interaction of the phosphorylated response regulator CheY with its phosphatase CheZ. Since this interaction defines the rate of CheY dephosphorylation, which at steady state equals the rate of CheY phosphorylation, it can be used to characterize intracellular kinase activity and thus to analyze properties of the chemotaxis signaling network.
Assuntos
Bactérias/metabolismo , Quimiotaxia , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Bactérias/metabolismo , Desenho de Equipamento , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Modelos Biológicos , Modelos Teóricos , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Probabilidade , Transdução de Sinais , Fatores de TempoRESUMO
Cellular networks are intrinsically subject to stochastic fluctuations, but analysis of the resulting noise remained largely limited to gene expression. The pathway controlling chemotaxis of Escherichia coli provides one example where posttranslational signaling noise has been deduced from cellular behavior. This noise was proposed to result from stochasticity in chemoreceptor methylation, and it is believed to enhance environment exploration by bacteria. Here we combined single-cell FRET measurements with analysis based on the fluctuation-dissipation theorem (FDT) to characterize origins of activity fluctuations within the chemotaxis pathway. We observed surprisingly large methylation-independent thermal fluctuations of receptor activity, which contribute to noise comparably to the energy-consuming methylation dynamics. Interactions between clustered receptors involved in amplification of chemotactic signals are also necessary to produce the observed large activity fluctuations. Our work thus shows that the high response sensitivity of this cellular pathway also increases its susceptibility to noise, from thermal and out-of-equilibrium processes.
Assuntos
Variação Biológica da População , Quimiotaxia , Escherichia coli/genética , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Transferência Ressonante de Energia de Fluorescência , Metilação , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Análise de Célula ÚnicaRESUMO
Internal epithelial surfaces in humans are both oxygenated and physically protected by a few hundred microns thick hydrogel mucosal layer, conditions that might support bacterial aerotaxis. However, the potential role of aerotaxis in crossing such a thin hydrogel layer is not clear. Here, we used a new setup to study the potential role of motility and chemotaxis in the bacterial colonization of surfaces covered by a thin hydrogel layer and subjected to a vertical oxygen gradient. Using the bacterium Escherichia coli, we show that both non-motile and motile-but-non-chemotactic bacteria could barely reach the surface. However, an acquired mutation in the non-chemotactic bacteria that altered their inherent swimming behavior led to a critical enhancement of surface colonization. Most chemotactic strains accumulated within the bulk of the hydrogel layer, except for the MG1655 strain, which showed a unique tendency to accumulate directly at the oxygenated surface and thus exhibited distinctly enhanced colonization. Even after a long period of bacterial growth, non-motile bacteria could not colonize the hydrogel. Thus, switching motility, which can be spontaneously acquired or altered in vivo, is critical for the colonization of such protected surfaces, whereas aerotaxis capacity clearly expedites surface colonization, and can lead to diverse colonization patterns.
Assuntos
Bactérias/crescimento & desenvolvimento , Quimiotaxia , Fenômenos Fisiológicos Bacterianos , Escherichia coli/fisiologia , Hidrogel de Polietilenoglicol-Dimetacrilato , Mutação , Propriedades de SuperfícieRESUMO
Motile bacteria use large receptor arrays to detect and follow chemical gradients in their environment. Extended receptor arrays, composed of networked signaling complexes, promote cooperative stimulus control of their associated signaling kinases. Here, we used structural lesions at the communication interface between core complexes to create an Escherichia coli strain with functional but dispersed signaling complexes. This strain allowed us to directly study how networking of signaling complexes affects chemotactic signaling and gradient-tracking performance. We demonstrate that networking of receptor complexes provides bacterial cells with about 10-fold-heightened detection sensitivity to attractants while maintaining a wide dynamic range over which receptor adaptational modifications can tune response sensitivity. These advantages proved especially critical for chemotaxis toward an attractant source under conditions in which bacteria are unable to alter the attractant gradient. IMPORTANCE: Chemoreceptor arrays are found in many motile bacteria. However, although our understanding of bacterial chemotaxis is quite detailed, the signaling and behavioral advantages of networked receptor arrays had not been directly studied in cells. We have recently shown that lesions in a key interface of the E. coli receptor array diminish physical connections and functional coupling between core signaling complexes while maintaining their basic signaling capacity. In this study, we exploited an interface 2 mutant to show, for the first time, that coupling between core complexes substantially enhances stimulus detection and chemotaxis performance.
Assuntos
Células Quimiorreceptoras/fisiologia , Quimiotaxia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Quimiotaxia/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Transdução de Sinais/genéticaRESUMO
Several bacterial sensory-kinase receptors form clusters on the cell membrane. However, the dynamics of sensory-kinase clustering are largely unclear. Using measurements of fluorescence anisotropy and time-lapse imaging of Escherichia coli cells, we demonstrate that copper ions trigger self-association of BaeS receptors and lead to rapid formation of clusters, which can be reversibly dispersed by a metal chelator. Copper ions did not trigger self-association of other fluorescently tagged sensory kinases, and other divalent metal ions could not elicit self-association of BaeS. The histidine residues in the BaeS periplasmic domain are essential for copper binding in vitro and are important for the copper-induced BaeS responses in vivo. BaeS clustering was triggered also under conditions that directly triggered BaeS-dependent transcriptional responses. Thus, clustering of sensory kinase receptors can be dynamic and context dependent and can be triggered by specific environmental cues.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas Quinases/metabolismo , Cobre/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina , Concentração de Íons de Hidrogênio , Mutação , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Proteínas Periplásmicas/genética , Regiões Promotoras Genéticas/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Transcrição Gênica/efeitos dos fármacos , Compostos de Tungstênio/farmacologiaRESUMO
Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfotransferases/metabolismo , Proteínas Quinases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Histidina Quinase , Fosfotransferases/genética , Proteínas Quinases/genéticaRESUMO
The bacterium Escherichia coli exhibits chemotactic behavior at temperatures ranging from approximately 20 °C to at least 42 °C. This behavior is controlled by clusters of transmembrane chemoreceptors made from trimers of dimers that are linked together by cross-binding to cytoplasmic components. By detecting fluorescence energy transfer between various components of this system, we studied the underlying molecular behavior of these receptors in vivo and throughout their operating temperature range. We reveal a sharp modulation in the conformation of unclustered and clustered receptor trimers and, consequently, in kinase activity output. These modulations occurred at a characteristic temperature that depended on clustering and were lower for receptors at lower adaptational states. However, in the presence of dynamic adaptation, the response of kinase activity to a stimulus was sustained up to 45 °C, but sensitivity notably decreased. Thus, this molecular system exhibits a clear thermal sensitivity that emerges at the level of receptor trimers, but both receptor clustering and adaptation support the overall robust operation of the system at elevated temperatures.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras/química , Células Quimiorreceptoras/metabolismo , Quimiotaxia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Temperatura Alta , Proteínas Luminescentes , Proteínas Quimiotáticas Aceptoras de Metil , Conformação Proteica , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , TermodinâmicaRESUMO
Bacterial chemoreceptors form mixed trimers of homodimers that cluster further in the presence of other cytoplasmic components. The physical proximity between receptors is thought to promote conformational coupling that enhances sensitivity, dynamic range, and collaboration between receptors of different types. We investigated conformational coupling between neighboring dimers by co-expressing two types of receptors, only one of which was labeled with yellow fluorescent protein. The two types of receptors were stimulated independently, and changes in the relative orientation of the labeled receptors were followed by fluorescence anisotropy. Possible coupling via cytoplasmic components of the taxis system was avoided by working with strains lacking those components. We find that binding of ligand to one type of receptor affects the conformation of the other type of receptor but not in the same way as binding of ligand to that receptor directly does. Thus, different receptors are coupled but not as simply as previously thought.
Assuntos
Bactérias , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Células Quimiorreceptoras/química , Células Quimiorreceptoras/metabolismo , Estrutura Quaternária de Proteína , Anisotropia , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/genética , Dimerização , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismoRESUMO
Two-component signaling systems play a major role in the long-term adaptation of microorganisms to changes in osmolarity, but how osmoreceptors work is not well understood. Temporal changes in solute concentration are sensed by the chemotaxis system in Escherichia coli, enabling these bacteria to avoid regions of high osmolarity. To study how osmolarity is detected in this system, we fused yellow fluorescent protein (YFP) to the C terminus of the serine or aspartate chemoreceptor, monitored the steady-state fluorescence polarization of YFP, and found that the polarization decreased substantially upon addition of osmotic agents. This decrease was due to an increase in fluorescence resonance energy transfer between YFP fluorophores in adjacent homodimers within trimers of dimers. Thus, changes in homodimer spacing and/or orientation appear to initiate osmotactic signaling.
Assuntos
Escherichia coli K12/metabolismo , Receptores de Aminoácido/metabolismo , Escherichia coli K12/genética , Polarização de Fluorescência , Genes Reporter , Mutação , Concentração Osmolar , Pressão Osmótica , Receptores de Aminoácido/genética , Estresse MecânicoRESUMO
Two-component signaling systems, in which a receptor-coupled kinase is used to control the phosphorylation level of a response regulator, are commonly used in bacteria to sense their environment. In the chemotaxis system of Escherichia coli, the receptors, and thus the kinase, are clustered on the inner cell membrane. The phosphatase of this system also is recruited to receptor clusters, but the reason for this association is not clear. By using FRET imaging of single cells, we show that in vivo the phosphatase activity is substantially larger at the cluster, indicating that the signaling source (the kinase) and the signaling sink (the phosphatase) tend to be located at the same place in the cell. When this association is disrupted, a gradient in the concentration of the phosphorylated response regulator appears, and the chemotactic response is degraded. Such colocalization is inevitable in systems in which the activity of the kinase and the phosphatase are produced by the same enzyme. Evidently, this design enables a more rapid and spatially uniform response.