RESUMEN
Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α-helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate-sink response regulator, CheY1~P by enhancing its decay two-fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 µM, which is 25-fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 µM, and a SEC-MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation.
RESUMEN
Hybrid two-component systems (HTCSs) comprise a major class of transcription regulators of polysaccharide utilization genes in Bacteroides. Distinct from classical two-component systems in which signal transduction is carried out by intermolecular phosphotransfer between a histidine kinase (HK) and a cognate response regulator (RR), HTCSs contain the membrane sensor HK and the RR transcriptional regulator within a single polypeptide chain. Tethering the DNA-binding domain (DBD) of the RR with the dimeric HK domain in an HTCS could potentially promote dimerization of the DBDs and would thus require a mechanism to suppress DNA-binding activity in the absence of stimulus. Analysis of phosphorylation and DNA-binding activities of several HTCSs from Bacteroides thetaiotaomicron revealed a DBD suppression mechanism in which an inhibitory interaction between the DBD and the phosphoryl group-accepting receiver domain (REC) decreases autophosphorylation rates of HTCS-RECs and represses DNA-binding activities in the absence of phosphorylation. Sequence analyses and structure predictions identified a highly conserved sequence motif correlated with a conserved inhibitory domain arrangement of REC and DBD. The presence of the motif, as in most HTCSs, or its absence, in a small subset of HTCSs, is likely predictive of two distinct regulatory mechanisms evolved for different glycans. Substitutions within the conserved motif relieve the inhibitory interaction and result in elevated DNA-binding activities in the absence of phosphorylation. Our data suggest a fundamental regulatory mechanism shared by most HTCSs to suppress DBD activities using a conserved inhibitory interdomain arrangement to overcome the challenge of the fused HK and RR components. IMPORTANCE: Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides, the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides.
Asunto(s)
Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Fosforilación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Unión Proteica , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Bacteroides/genética , Bacteroides/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/química , Dominios Proteicos , Transducción de SeñalRESUMEN
Binding of transcription factor (TF) proteins to regulatory DNA sites is key to accurate control of gene expression in response to environmental stimuli. Theoretical modeling of transcription regulation is often focused on a limited set of genes of interest, while binding of the TF to other genomic sites is seldom considered. The total number of TF binding sites (TFBSs) affects the availability of TF protein molecules and sequestration of a TF by TFBSs can promote bistability. For many signaling systems where a graded response is desirable for continuous control over the input range, biochemical parameters of the regulatory proteins need be tuned to avoid bistability. Here we analyze the mono-/bistable parameter range for positively autoregulated two-component systems (TCSs) in the presence of different numbers of competing TFBSs. TCS signaling, one of the major bacterial signaling strategies, couples signal perception with output responses via protein phosphorylation. For bistability, competition for TF proteins by TFBSs lowers the requirement for high fold change of the autoregulated transcription but demands high phosphorylation activities of TCS proteins. We show that bistability can be avoided with a low phosphorylation capacity of TCSs, a high TF affinity for the autoregulated promoter or a low fold change in signaling protein levels upon induction. These may represent general design rules for TCSs to ensure uniform graded responses. Examining the mono-/bistability parameter range allows qualitative prediction of steady-state responses, which are experimentally validated in the E. coli CusRS system.
Asunto(s)
Factores de Transcripción , Sitios de Unión , Escherichia coli/genética , Transducción de Señal , Unión Proteica , Estabilidad ProteicaRESUMEN
Pathogenic bacteria can rapidly respond to stresses such as reactive oxygen species (ROS) using reversible redox-sensitive oxidation of cysteine thiol (-SH) groups in regulators. Here, we use proteomics to profile reversible ROS-induced thiol oxidation in Vibrio cholerae, the etiologic agent of cholera, and identify two modified cysteines in ArcA, a regulator of global carbon oxidation that is phosphorylated and activated under low oxygen. ROS abolishes ArcA phosphorylation but induces the formation of an intramolecular disulfide bond that promotes ArcA-ArcA interactions and sustains activity. ArcA cysteines are oxidized in cholera patient stools, and ArcA thiol oxidation drives in vitro ROS resistance, colonization of ROS-rich guts, and environmental survival. In other pathogens, such as Salmonella enterica, oxidation of conserved cysteines of ArcA orthologs also promotes ROS resistance, suggesting a common role for ROS-induced ArcA thiol oxidation in modulating ArcA activity, allowing for a balance of expression of stress- and pathogenesis-related genetic programs.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Cólera/microbiología , Proteoma/metabolismo , Proteínas Represoras/metabolismo , Salmonella enterica/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Vibrio cholerae/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Cisteína/metabolismo , Heces/microbiología , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Proteómica/métodos , Especies Reactivas de Oxígeno/metabolismo , Infecciones por Salmonella/microbiología , Vibrio cholerae/genéticaRESUMEN
Studies of transcription regulation are often focused on binding of transcription factors (TFs) to a small number of promoters of interest. It is often assumed that TFs are in great excess to their binding sites (TFBSs) and competition for TFs between DNA sites is seldom considered. With increasing evidence that TFBSs are exceedingly abundant for many TFs and significant variations in TF and TFBS numbers occur during growth, the interplay between a TF and all TFBSs should not be ignored. Here, we use additional decoy DNA sites to quantitatively analyze how the relative abundance of a TF to its TFBSs impacts the steady-state level and onset time of gene expression for the auto-activated Escherichia coli PhoB response regulator. We show that increasing numbers of decoy sites progressively delayed transcription activation and lowered promoter activities. Perturbation of transcription regulation by additional TFBSs did not require extreme numbers of decoys, suggesting that PhoB is approximately at capacity for its DNA sites. Addition of decoys also converted a graded response to a bi-modal response. We developed a binding competition model that captures the major features of experimental observations, providing a quantitative framework to assess how variations in TFs and TFBSs influence transcriptional responses.
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ADN/química , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Motivos de Nucleótidos , Factores de Transcripción/metabolismo , Sitios de Unión , ADN/genética , ADN/metabolismo , Escherichia coli , Proteínas de Escherichia coli/química , Unión Proteica , Factores de Transcripción/químicaRESUMEN
Histidine protein kinases (HKs) are prevalent prokaryotic sensor kinases that are central to phosphotransfer in two-component signal transduction systems, regulating phosphorylation of response regulator proteins that determine the output responses. HKs typically exist as dimers and can potentially autophosphorylate at each conserved histidine residue in the individual protomers, leading to diphosphorylation. However, analyses of HK phosphorylation in biochemical assays in vitro suggest negative cooperativity, whereby phosphorylation in one protomer of the dimer inhibits phosphorylation in the second protomer, leading to â¼50% phosphorylation of the available sites in dimers. This negative cooperativity is often correlated with an asymmetric domain arrangement, a common structural characteristic of autophosphorylation states in many HK structures. In this study, we engineered covalent dimers of the cytoplasmic domains of Escherichia coli CpxA, enabling us to quantify individual species: unphosphorylated, monophosphorylated, and diphosphorylated dimers. Together with mathematical modeling, we unambiguously demonstrate no cooperativity in autophosphorylation of CpxA despite its asymmetric structures, indicating that these asymmetric domain arrangements are not linked to negative cooperativity and hemiphosphorylation. Furthermore, the modeling indicated that many parameters, most notably minor amounts of ADP generated during autophosphorylation reactions or present in ATP preparations, can produce â¼50% total phosphorylation that may be mistakenly attributed to negative cooperativity. This study also establishes that the engineered covalent heterodimer provides a robust experimental system for investigating cooperativity in HK autophosphorylation and offers a useful tool for testing how symmetric or asymmetric structural features influence HK functions.
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Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Adenosina Difosfato/análisis , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/análisis , Adenosina Trifosfatasas/metabolismo , Modelos Moleculares , Fosforilación , Conformación ProteicaRESUMEN
Although it has long been known that bacteria detect and react to plant chemicals to establish an interaction, the cellular signaling mechanisms involved in these perception processes have hitherto remained obscure. Some exciting recent advances in the field have described, for the first time, how some phytopathogenic bacteria sense the host plant hormones, cytokinins. These discoveries not only advance the understanding of cell signaling circuitries engaged in cytokinin sensing in non-plant organisms, but also increase our knowledge of the broad role of these ancient molecules in regulating intra- and interspecific communications.
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Bacterias/genética , Fenómenos Fisiológicos Bacterianos/genética , Citocininas/química , Reguladores del Crecimiento de las Plantas/química , Citocininas/metabolismo , Regulación de la Expresión Génica de las Plantas , Plantas/química , Plantas/microbiología , Transducción de Señal/genéticaRESUMEN
Originally known for their roles in a myriad of bacterial processes, the two-component signaling systems are now found far beyond the bacterial domain. Papon and Stock highlight the many interesting features of these widespread signaling systems.
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Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Genoma Microbiano , Transducción de Señal , Evolución Molecular , Dominios y Motivos de Interacción de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
In response to the increasingly problematic emergence of antibiotic resistance, novel strategies for combating pathogenic bacteria are being investigated. Targeting the agr quorum sensing system, which regulates expression of virulence in Staphylococcus aureus, is one potentially useful approach for combating drug-resistant pathogens that has not yet been fully explored. A previously published study of a fragment screen resulted in the identification of five compound fragments that interact with the DNA-binding domain of the response regulator AgrA from S. aureus. We have analyzed the ability of these compounds to affect agr-mediated virulence gene expression in cultured S. aureus cells. Three of the compounds demonstrated the ability to reduce agr-driven transcription at the P2 and P3 promoters of the agr operon and increase biofilm formation, and two of these compounds also showed the ability to reduce levels of secreted toxins. The finding that the compounds tested were able to reduce agr activity suggests that they could be useful tools for probing the effects of agr inhibition. Furthermore, the characteristics of compound fragments make them good starting materials for the development of compound libraries to iteratively improve the inhibitors.
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Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Hemólisis/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Transactivadores/metabolismo , Virulencia/efectos de los fármacos , Animales , Proteínas Bacterianas/genética , Operón , Regiones Promotoras Genéticas , Percepción de Quorum , Conejos , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Transactivadores/genética , Factores de VirulenciaRESUMEN
Response regulators function as the output components of two-component systems, which couple the sensing of environmental stimuli to adaptive responses. Response regulators typically contain conserved receiver (REC) domains that function as phosphorylation-regulated switches to control the activities of effector domains that elicit output responses. This modular design is extremely versatile, enabling different regulatory strategies tuned to the needs of individual signaling systems. This review summarizes structural features that underlie response regulator function. An abundance of atomic resolution structures and complementary biochemical data have defined the mechanisms for response regulator enzymatic activities, revealed trends in regulatory strategies utilized by response regulators of different subfamilies, and provided insights into interactions of response regulators with their cognate histidine kinases. Among the hundreds of thousands of response regulators identified, variations abound. This article provides a framework for understanding structural features that enable function of canonical response regulators and a basis for distinguishing noncanonical configurations.
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Histidina Quinasa/química , Histidina Quinasa/metabolismo , Transducción de Señal , Conformación Proteica , Dominios ProteicosAsunto(s)
Experimentación Animal/ética , Arabidopsis/genética , Biología/tendencias , Farmacorresistencia Bacteriana/genética , Publicaciones Periódicas como Asunto , Receptores de Antígenos de Linfocitos T/genética , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Antibacterianos/farmacología , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Transporte Biológico , Quirópteros/fisiología , Vesículas Cubiertas por Clatrina/inmunología , Vesículas Cubiertas por Clatrina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Endocitosis/inmunología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Vuelo Animal/fisiología , Humanos , Feromonas/genética , Feromonas/metabolismo , Conformación Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Signal transduction systems configured around a core phosphotransfer step between a histidine kinase and a cognate response regulator protein occur in organisms from all domains of life. These systems, termed two-component systems, constitute the majority of multi-component signaling pathways in Bacteria but are less prevalent in Archaea and Eukarya. The core signaling domains are modular, allowing versatility in configuration of components into single-step phosphotransfer and multi-step phosphorelay pathways, the former being predominant in bacteria and the latter in eukaryotes. Two-component systems regulate key cellular regulatory processes that provide adaptive responses to environmental stimuli and are of interest for the development of antimicrobial therapeutics, biotechnology applications, and biosensor engineering. In bacteria, two-component systems have been found to mediate responses to an extremely broad array of extracellular and intracellular chemical and physical stimuli, whereas in archaea and eukaryotes, the use of two-component systems is more limited. This review summarizes recent advances in exploring the repertoire of sensor histidine kinases in the Archaea and Eukarya domains of life.
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Archaea , Eucariontes , Histidina Quinasa , Archaea/enzimología , Eucariontes/enzimología , Histidina , Transducción de SeñalRESUMEN
A fundamental trade-off between rapid response and optimal expression of genes below cytotoxic levels exists for many signaling circuits, particularly for positively autoregulated systems with an inherent response delay. Here, we describe a regulatory scheme in the E. coli PhoB-PhoR two-component system, which overcomes the cost of positive feedback and achieves both fast and optimal steady-state response for maximal fitness across different environments. Quantitation of the cellular activities enables accurate modeling of the response dynamics to describe how requirements for optimal protein concentrations place limits on response speed. An observed fast response that exceeds the limit led to the prediction and discovery of a coupled negative autoregulation, which allows fast gene expression without increasing steady-state levels. We demonstrate the fitness advantages for the coupled feedbacks in both dynamic and stable environments. Such regulatory schemes offer great flexibility for accurate control of gene expression levels and dynamics upon environmental changes.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Homeostasis/fisiología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Homeostasis/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Quantitation of two-component protein activities is becoming increasingly important to understand the general design principles for this widely distributed prokaryotic signaling pathway. In many two-component systems (TCSs), phosphatase activity of the sensor histidine kinase (HK) plays a major role in controlling the system output and resetting the system to the prestimulus state. Quantitation of the phosphatase activity is often carried out in vitro, usually with truncated proteins that may not recapitulate the intact HK in the cellular environment. This chapter outlines a method for characterizing the intracellular phosphatase activity by investigating the TCS deactivation dynamics upon stimulus withdrawal. Two experimental approaches, the direct Phos-tag gel analysis and the indirect reporter assay, are described for measuring the TCS deactivation dynamics in cell. Combined with a mathematic model, the experimentally determined kinetics can lead to proper evaluation of the intracellular phosphatase activity.
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Citoplasma/metabolismo , Pruebas de Enzimas/métodos , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Histidina Quinasa/metabolismo , Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Pruebas de Enzimas/instrumentación , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Histidina Quinasa/química , Cinética , Modelos Biológicos , FosforilaciónRESUMEN
Fluctuations in nutrient availability often result in recurrent exposures to the same stimulus conditions. The ability to memorize the past event and use the "memory" to make adjustments to current behaviors can lead to a more efficient adaptation to the recurring stimulus. A short-term phenotypic memory can be conferred via carryover of the response proteins to facilitate the recurrent response, but the additional accumulation of response proteins can lead to a deviation from response homeostasis. We used the Escherichia coli PhoB/PhoR two-component system (TCS) as a model system to study how cells cope with the recurrence of environmental phosphate (Pi) starvation conditions. We discovered that "memory" of prior Pi starvation can exert distinct effects through two regulatory pathways, the TCS signaling pathway and the stress response pathway. Although carryover of TCS proteins can lead to higher initial levels of transcription factor PhoB and a faster initial response in prestarved cells than in cells not starved, the response enhancement can be overcome by an earlier and greater repression of promoter activity in prestarved cells due to the memory of the stress response. The repression counterbalances the carryover of the response proteins, leading to a homeostatic response whether or not cells are prestimulated. A computational model based on sigma factor competition was developed to understand the memory of stress response and to predict the homeostasis of other PhoB-regulated response proteins. Our insight into the history-dependent PhoBR response may provide a general understanding of how TCSs respond to recurring stimuli and adapt to fluctuating environmental conditions.IMPORTANCE Bacterial cells in their natural environments experience scenarios that are far more complex than are typically replicated in laboratory experiments. The architectures of signaling systems and the integration of multiple adaptive pathways have evolved to deal with such complexity. In this study, we examined the molecular "memory" that is generated by previous exposure to stimulus. Under our experimental conditions, activating effects of autoregulated two-component signaling and inhibitory effects of the stress response counterbalanced the transcriptional output to approach response homeostasis whether or not cells had been preexposed to stimulus. Modeling allows prediction of response behavior in different scenarios and demonstrates both the robustness of the system output and its sensitivity to historical parameters such as timing and levels of exposure to stimuli.
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Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Transducción de Señal , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Teóricos , Fosfatos/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismoRESUMEN
Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo, which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression.IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.