Two-component Signal Transduction Pathway Research Articles

The cytoplasmic phosphate level has a central regulatory role in the phosphate starvation response of Caulobacter crescentus

In bacteria, the availability of environmental inorganic phosphate is typically sensed by the conserved PhoR-PhoB two-component signal transduction pathway, which uses the flux through the PstSCAB phosphate transporter as a readout of the extracellular phosphate level to control phosphate-responsive genes. While the sensing of environmental phosphate is well-investigated, the regulatory effects of cytoplasmic phosphate are unclear. Here, we disentangle the physiological and transcriptional responses of Caulobacter crescentus to changes in the environmental and cytoplasmic phosphate levels by uncoupling phosphate uptake from the activity of the PstSCAB system, using an additional, heterologously produced phosphate transporter. This approach reveals a two-pronged response of C. crescentus to phosphate limitation, in which PhoR-PhoB signaling mostly facilitates the utilization of alternative phosphate sources, whereas the cytoplasmic phosphate level controls the morphological and physiological adaptation of cells to growth under global phosphate limitation. These findings open the door to a comprehensive understanding of phosphate signaling in bacteria.

Development of a Light-Dependent Protein Histidine Kinase.

Phosphorylation plays a critical role in facilitating signal transduction in prokaryotic and eukaryotic organisms. Our study introduces a tool for investigation of signal diffusion in a biochemical regulation network through the design and characterization of a light-stimulated histidine kinase that consists of the LOV domain from YtvA from Bacillus subtilis and the histidine kinase domain Sln1 from Saccharomyces cerevisiae. We show that blue light can be used as a trigger for modulation of the phosphorylation events in this engineered two-component signal transduction pathway in a eukaryotic cell. At the same time, we demonstrate the robustness of LOV domains and their utility for designing fusion proteins for signal transduction that can be triggered with (blue) light, providing a ready toolkit to design blue light dependent two-component signalling pathways.

Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria

BackgroundBacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons. MethodsElectron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR. ResultsAlterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between QA and cytochrome b6f, with reduction favoring CrhR accumulation. ConclusionsThe results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of QB/PQH2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression. General significanceThe potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated.

Progress in regulatory mechanism for inducing β-lactamase in Gram-negative bacteria

Beta-lactams are the most widely used antibiotics. One of the principle mechanisms for Gram-negative bacteria to resist β-lactams is by producing β-lactamases that degrade β-lactams. This review highlights two regulatory mechanisms for inducing β-lactamase in Gram-negative bacteria. In the ampR-ampC paradigm, the induction of β-lactamase is intimately linked to peptidoglycan recycling. AmpR, a LysR-type transcriptional regulator, plays a central role in regulating expression of β-lactamase. Recent studies found that two-component signal transduction pathway is activated by β-lactams, which in turn induces the expression of β-lactamase. Finally, we discussed the future research directions in β-lactam resistance in Gram-negative bacteria.

Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae.

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation, and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well-studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation.

The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

Cross-talk and specificity in two-component signal transduction pathways.

Two-component signaling systems (TCSs) are composed of two proteins, sensor kinases and response regulators, which can cross-talk and integrate information between them by virtue of high-sequence conservation and modular nature, to generate concerted and diversified responses. However, TCSs have been shown to be insulated, to facilitate linear signal transmission and response generation. Here, we discuss various mechanisms that confer specificity or cross-talk among TCSs. The presented models are supported with evidence that indicate the physiological significance of the observed TCS signaling architecture. Overall, we propose that the signaling topology of any TCSs cannot be predicted using obvious sequence or structural rules, as TCS signaling is regulated by multiple factors, including spatial and temporal distribution of the participating proteins.

SepM, a Streptococcal Protease Involved in Quorum Sensing, Displays Strict Substrate Specificity.

Streptococcus mutans, a causative agent of dental caries, relies on multiple quorum-sensing (QS) pathways that coordinate the expression of factors needed for colonization in the oral cavity. S. mutans uses small peptides as QS signaling molecules that typically are secreted into the outside milieu. Competence-stimulating peptide (CSP) is one such QS signaling molecule that functions through the ComDE two-component signal transduction pathway. CSP is secreted through NlmTE, a dedicated ABC transporter that cleaves off the N-terminal leader peptide to generate a mature peptide that is 21 residues long (CSP-21). We recently identified a surface-localized protease, SepM, which further cleaves the CSP-21 peptide at the C-terminal end and removes the last 3 residues to generate CSP-18. CSP-18 is the active QS molecule that interacts with the ComD sensor kinase to activate the QS pathway. In this study, we show that SepM specifically cleaves CSP-21 between the Ala18 and Leu19 residues. We also show that SepM recognizes only Ala at position 18 and Leu at position 19, although some CSP-18 variants with a substitution at position 18 can function equally as well as the QS peptide. Furthermore, we demonstrate that SepM homologs from other streptococci are capable of processing CSP-21 to generate functional CSP-18. SepM is a membrane-associated streptococcal protease that processes competence-stimulating peptide (CSP) to generate an active quorum-sensing molecule in S. mutans. SepM belongs to the S16 family of serine proteases, and in this study, we found that SepM behaves as an endopeptidase. SepM displays strict substrate specificity and cleaves the peptide bond between the Ala and Leu residues. This is the first report of an endopeptidase that specifically cleaves these two residues.

Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa.

Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P. aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments.

Reverse engineering of logic-based differential equation models using a mixed-integer dynamic optimization approach.

Motivation: Systems biology models can be used to test new hypotheses formulated on the basis of previous knowledge or new experimental data, contradictory with a previously existing model. New hypotheses often come in the shape of a set of possible regulatory mechanisms. This search is usually not limited to finding a single regulation link, but rather a combination of links subject to great uncertainty or no information about the kinetic parameters.Results: In this work, we combine a logic-based formalism, to describe all the possible regulatory structures for a given dynamic model of a pathway, with mixed-integer dynamic optimization (MIDO). This framework aims to simultaneously identify the regulatory structure (represented by binary parameters) and the real-valued parameters that are consistent with the available experimental data, resulting in a logic-based differential equation model. The alternative to this would be to perform real-valued parameter estimation for each possible model structure, which is not tractable for models of the size presented in this work. The performance of the method presented here is illustrated with several case studies: a synthetic pathway problem of signaling regulation, a two-component signal transduction pathway in bacterial homeostasis, and a signaling network in liver cancer cells.Supplementary information: Supplementary data are available at Bioinformatics online.Contact: julio@iim.csic.es or saezrodriguez@ebi.ac.uk

Full-length structure of a monomeric histidine kinase reveals basis for sensory regulation.

Although histidine kinases (HKs) are critical sensors of external stimuli in prokaryotes, the mechanisms by which their sensor domains control enzymatic activity remain unclear. Here, we report the full-length structure of a blue light-activated HK from Erythrobacter litoralis HTCC2594 (EL346) and the results of biochemical and biophysical studies that explain how it is activated by light. Contrary to the standard view that signaling occurs within HK dimers, EL346 functions as a monomer. Its structure reveals that the light-oxygen-voltage (LOV) sensor domain both controls kinase activity and prevents dimerization by binding one side of a dimerization/histidine phosphotransfer-like (DHpL) domain. The DHpL domain also contacts the catalytic/ATP-binding (CA) domain, keeping EL346 in an inhibited conformation in the dark. Upon light stimulation, interdomain interactions weaken to facilitate activation. Our data suggest that the LOV domain controls kinase activity by affecting the stability of the DHpL/CA interface, releasing the CA domain from an inhibited conformation upon photoactivation. We suggest parallels between EL346 and dimeric HKs, with sensor-induced movements in the DHp similarly remodeling the DHp/CA interface as part of activation.

Two-Component Histidine Phosphotransfer Protein Ypd1 Is Not Essential for Viability in Candida albicans

Prokaryotes and lower eukaryotes, such as yeasts, utilize two-component signal transduction pathways to adapt cells to environmental stress and to regulate the expression of genes associated with virulence. One of the central proteins in this type of signaling mechanism is the phosphohistidine intermediate protein Ypd1. Ypd1 is reported to be essential for viability in the model yeast Saccharomyces cerevisiae. We present data here showing that this is not the case for Candida albicans. Disruption of YPD1 causes cells to flocculate and filament constitutively under conditions that favor growth in yeast form. To determine the function of Ypd1 in the Hog1 mitogen-activated protein kinase (MAPK) pathway, we measured phosphorylation of Hog1 MAPK in ypd1Δ/Δ and wild-type strains of C. albicans. Constitutive phosphorylation of Hog1 was observed in the ypd1Δ/Δ strain compared to the wild-type strain. Furthermore, fluorescence microscopy revealed that green fluorescent protein (GFP)-tagged Ypd1 is localized to both the nucleus and the cytoplasm. The subcellular segregation of GFP-tagged Ypd1 hints at an important role(s) of Ypd1 in regulation of Ssk1 (cytosolic) and Skn7 (nuclear) response regulator proteins via phosphorylation in C. albicans. Overall, our findings have profound implications for a mechanistic understanding of two-component signaling pathways in C. albicans, and perhaps in other pathogenic fungi.

Genetic Proclivities of Two-Component Modulated Aerobiosis

Great advances have been made in the past five decades in understanding the molecular mechanics of the two-component signal transduction pathway in bacteria but its applications in Medicine and Food Industries are yet to be fully unravelled. We discuss the varying changes in the extracellular environment of bacteria and their possession of multiple Two-Component Systems with each being specialize to react to a specific environmental signal, such as pH, nutrient level, redox state, osmotic pressure, quorum signals, and antibiotics. The sensitivity of this response transmits information between different Two-Component Systems to form a complex signal transduction network. Bacteria’s signal transduction system, referred to as a two-component system, are essential for adaptation to external stimuli. These systems provides a signal transduction pathways widely employed from prokaryotes to eukaryotes. Typically, each two-component system composed of a sensor protein distinctively monitors an external signal(s) and a response regulator (RR) that controls gene expression and other physiological activities which are collectively assembled in a signal transduction pathway. This annex reviews the molecular mechanics underlying the signal transduction systems in prokaryotic organisms. It is not uncommon to hear, either explicitly or implicitly, the statement that “two component regulatory systems are well understood”. Therefore, we examine the current models of the mechanisms of the regulatory systems and provide viable suggestions to further expand its applications in drug efficiency and antibiotic resistance in humans as well as enhancing the shelf-life of products in the food industry. We also outline the challenges that might have quenched possible trials of this application to human health.

Genome-Wide Identification, Phylogeny, Evolution and Expression Patterns of AP2/ERF Genes and Cytokinin Response Factors in Brassica rapa ssp. pekinensis

The AP2/ERF transcription factor family is one of the largest families involved in growth and development, hormone responses, and biotic or abiotic stress responses in plants. In this study, 281 AP2/ERF transcription factor unigenes were identified in Chinese cabbage. These superfamily members were classified into three families (AP2, ERF, and RAV). The ERF family was subdivided into the DREB subfamily and the ERF subfamily with 13 groups (I– XI) based on sequence similarity. Duplication, evolution and divergence of the AP2/ERF genes in B. rapa and Arabidopsis thaliana were investigated and estimated. Cytokinin response factors (CRFs), as a subclade of the AP2/ERF family, are important transcription factors that define a branch point in the cytokinin two-component signal (TCS) transduction pathway. Up to 21 CRFs with a conserved CRF domain were retrieved and designated as BrCRFs. The amino acid sequences, conserved regions and motifs, phylogenetic relationships, and promoter regions of the 21 BrCRFs were analyzed in detail. The BrCRFs broadly expressed in various tissues and organs. The transcripts of BrCRFs were regulated by factors such as drought, high salinity, and exogenous 6-BA, NAA, and ABA, suggesting their involvement in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. These results provide new insight into the divergence, variation, and evolution of AP2/ERF genes at the genome-level in Chinese cabbage.

Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways

The histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the Saccharomyces cerevisiae multistep two-component signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both S. cerevisiae and in Cryptococcus neoformans. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in S. cerevisiae, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on S. cerevisiae Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.

Helix Bundle Loops Determine Whether Histidine Kinases Autophosphorylate in cis or in trans

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.

Computational and Experimental Analyses Reveal the Essential Roles of Interdomain Linkers in the Biological Function of Chemotaxis Histidine Kinase CheA

A two-component signal transduction pathway underlies the phenomenon of bacterial chemotaxis that allows bacteria to modulate their swimming behavior in response to environmental stimuli. The dimeric five-domain histidine kinase, CheA, plays a central role in the pathway, converting sensory signals to a chemical signal via trans-autophosphorylation between the P1 and P4 domains. This autophosphorylation is regulated via the networked interactions among the P5 domain of CheA, CheW, and chemoreceptors. Despite a wealth of structural information about these components and their interactions, the key question of how the kinase activity of the catalytic P4 domain is regulated by the signal received from the regulatory P5 domain remains poorly understood. We performed replica exchange molecular dynamics simulations on the CheA kinase core and found that while individual domains maintained their structural fold, these domains exhibited a variety of interdomain orientations due to two interdomain linkers. A partially populated conformation that adopts an interdomain arrangement is suitable for building a functional ternary complex. An allosteric network derived from this structural model implies critical roles for two linkers in CheA's activity. The biochemical and biological functions of these linkers were assigned via a series of biochemical and genetic assays that show the P4-P5 linker controls the activation of CheA and the P3-P4 linker controls both the basal autophosphorylation activity and the activation of CheA. These results reveal the functional dependence between the two linkers and the essential role of the linkers in passing signal information from one domain to another.

An extracelluar protease, SepM, generates functional competence-stimulating peptide in Streptococcus mutans UA159.

Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans.

A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals

Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas.

Evolution of Two-Component Signal Transduction Systems

To exist in a wide range of environmental niches, bacteria must sense and respond to a variety of external signals. A primary means by which this occurs is through two-component signal transduction pathways, typically composed of a sensor histidine kinase that receives the input stimuli and then phosphorylates a response regulator that effects an appropriate change in cellular physiology. Histidine kinases and response regulators have an intrinsic modularity that separates signal input, phosphotransfer, and output response; this modularity has allowed bacteria to dramatically expand and diversify their signaling capabilities. Recent work has begun to reveal the molecular basis by which two-component proteins evolve. How and why do orthologous signaling proteins diverge? How do cells gain new pathways and recognize new signals? What changes are needed to insulate a new pathway from existing pathways? What constraints are there on gene duplication and lateral gene transfer? Here, we review progress made in answering these questions, highlighting how the integration of genome sequence data with experimental studies is providing major new insights.