蓝藻的二元信号传导系统

二元系统是细菌中主要的信号传导途径 ,磷酸根转移介导的信号途径使细胞得以感受各种环境刺激并产生应答。组氨酸蛋白质激酶的自动磷酸化将磷酸基团传给反应调节蛋白 ,反过来作为分子开关控制不同的效应物活性。蓝藻是地球上最早出现的光合自养原核生物 ,在长期的生物进化过程中 ,它们发展了一系列独特的形态和生理代谢机制 ,使其能在各种不同生境中生长、繁殖和扩增。研究蓝藻信号传导途径为阐明其高度的环境适应性提供了理论基础。     

Role of two-component regulatory systems in intracellular survival of Mycobacterium tuberculosis

The typical two-component regulatory systems (TCSs), consisting of response regulator and histidine kinase, play a central role in survival of pathogenic bacteria under stress conditions such as nutrient starvation, hypoxia, and nitrosative stress. A total of 11 complete paired two-component regulatory systems have been found in Mycobacterium tuberculosis, including a few isolated kinase and regulatory genes. Increasing evidence has shown that TCSs are closely associated with multiple physiological process like intracellular persistence, pathogenicity, and metabolism. This review gives the two-component signal transduction systems in M. tuberculosis and their signal transduction roles in adaption to the environment.     

Inhibition of the signal transduction through the AtoSC system by histidine kinase inhibitors in Escherichia coli

AtoSC two-component system participates in many indispensable processes of Escherichia coli. We report here that the AtoSC signal transduction is inhibited by established histidine kinase inhibitors. Closantel, RWJ-49815 and TNP-ATP belonging to different chemical classes of inhibitors, abrogated the in vitro AtoS kinase autophosphorylation. However, when AtoS was embedded in the membrane fractions, higher inhibitor concentrations were required for total inhibition. When AtoS interacted with AtoC forming complex, the intrinsic histidine kinase was protected by the response regulator, requiring increased inhibitors concentrations for partially AtoS autophosphorylation reduction. The inhibitors exerted an additional function on AtoSC, blocking the phosphotransfer from AtoS to AtoC, without however, affecting AtoC~P dephosphorylation. Their in vivo consequences through the AtoSC inhibition were elucidated on atoDAEB operon expression, which was inhibited only in AtoSC-expressing bacteria where AtoSC was induced by acetoacetate or spermidine. The inhibitor effects were extended on the AtoSC regulatory role on cPHB [complexed poly-(R)-3-hydroxybutyrate] biosynthesis. cPHB was decreased upon the blockers only in acetoacetate-induced AtoSC-expressing cells. Increased ATP amounts during bacterial growth reversed the inhibitory TNP-ATP-mediated effect on cPHB. The alteration of pivotal E. coli processes as an outcome of AtoSC inhibition, establish this system as a target of two-component systems inhibitors.     

Mammalian histidine kinases

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.     

Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.     

Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes Bacteria

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria.     

Bacterial signal transduction networks via connectors and development of the inhibitors as alternative antibiotics

Bacterial cells possess a signal transduction system that differs from those described in higher organisms, including human cells. These so-called two-component signal transduction systems (TCSs) consist of a sensor (histidine kinase, HK) and a response regulator, and are involved in cellular functions, such as virulence, drug resistance, biofilm formation, cell wall synthesis, cell division. They are conserved in bacteria across all species. Although TCSs are often studied and characterized individually, they are assumed to interact with each other and form signal transduction networks within the cell. In this review, I focus on the formation of TCS networks via connectors. I also explore the possibility of using TCS inhibitors, especially HK inhibitors, as alternative antimicrobial agents.     

Bacterial observations: a rudimentary form of intelligence?

Genome sequencing has revealed that signal transduction in bacteria makes use of a limited number of different devices, such as two-component systems, LuxI-LuxR quorum-sensing systems, phosphodiesterases, Ser-Thr (serine-threonine) kinases, OmpR-type regulators, and sigma factor-anti-sigma factor pathways. These systems use modular proteins with a large variety of input and output domains, yet strikingly conserved transmission domains. This conservation might lead to redundancy of output function, for example, via crosstalk (i.e. phosphoryl transfer from a non-cognate sensory kinase). The number of similar devices in a single cell, particularly of the two-component type, might amount to several dozen, and most of these operate in parallel. This could bestow bacteria with cellular intelligence if the network of two-component systems in a single cell fulfils the requirements of a neural network. Testing these ideas poses a great challenge for prokaryotic systems biology.     

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.     

Repurposing Hsp90 inhibitors as antibiotics targeting histidine kinases

To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.     

Dynamic Interaction between the CpxA Sensor Kinase and the Periplasmic Accessory Protein CpxP Mediates Signal Recognition in E. coli

Two-component systems, consisting of an inner membrane sensor kinase and a cytosolic response regulator, allow bacteria to respond to changes in the environment. Some two-component systems are additionally orchestrated by an accessory protein that integrates additional signals. It is assumed that spatial and temporal interaction between an accessory protein and a sensor kinase modifies the activity of a two-component system. However, for most accessory proteins located in the bacterial envelope the mechanistic details remain unclear. Here, we analyzed the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in Escherichia coli in dependency of three specific stimuli. The Cpx two-component system responds to envelope stress and plays a pivotal role for the quality control of multisubunit envelope structures, including type three secretion systems and pili of different pathogens. In unstressed cells, CpxP shuts off the Cpx response by a yet unknown mechanism. We show for the first time the physical interaction between CpxP and CpxA in unstressed cells using bacterial two-hybrid system and membrane-Strep-tagged protein interaction experiments. In addition, we demonstrate that a high salt concentration and the misfolded pilus subunit PapE displace CpxP from the sensor kinase CpxA in vivo. Overall, this study provides clear evidence that CpxP modulates the activity of the Cpx system by dynamic interaction with CpxA in response to specific stresses.     

Comprehensive DNA microarray analysis of Bacillus subtilis two-component regulatory systems.

It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B. subtilis two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (http://www.genome.ad.jp/kegg/expression).     

Characterization of the BaeSR two-component system from Salmonella Typhimurium and its role in ciprofloxacin-induced mdtA expression

Two-component systems are one of the most prevalent mechanisms by which bacteria sense, respond and adapt to changes in their environment. The activation of a sensor histidine kinase leads to autophosphorylation of a conserved histidine residue followed by transfer of the phosphoryl group to a cognate response regulator in an aspartate residue. The search for antibiotics that inhibit molecular targets has led to study prokaryotic two-component systems. In this study, we characterized in vitro and in vivo the BaeSR two-component system from Salmonella Typhimurium and evaluated its role in mdtA regulation in response to ciprofloxacin treatment. We demonstrated in vitro that residue histidine 250 is essential for BaeS autophosphorylation and aspartic acid 61 for BaeR transphosphorylation. By real-time PCR, we showed that mdtA activation in the presence of ciprofloxacin depends on both members of this system and that histidine 250 of BaeS and aspartic acid 61 of BaeR are needed for this. Moreover, the mdtA expression is directly regulated by binding of BaeR at the promoter region, and this interaction is enhanced when the protein is phosphorylated. In agreement, a BaeR mutant unable to phosphorylate at aspartic acid 61 presents a lower affinity with the mdtA promoter.     

Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems

Two-component systems are the most common mechanism of transmembrane signal transduction in bacteria. A typical system consists of a histidine kinase and a partner response regulator. The histidine kinase senses an environmental signal, which it transmits to its partner response regulator via a series of autophosphorylation, phosphotransfer, and dephosphorylation reactions. Much work has been done on particular systems, including several systems with regulatory roles in cellular physiology, communication, development, and, in the case of bacterial pathogens, the expression of genes important for virulence. We used two methods to investigate two-component regulatory systems in Escherichia coli K-12. First, we systematically constructed mutants with deletions of all two-component systems by using a now-standard technique of gene disruption (K. A. Datsenko and B. L. Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). We then analyzed these deletion mutants with a new technology called Phenotype MicroArrays, which permits assays of nearly 2,000 growth phenotypes simultaneously. In this study we tested 100 mutants, including mutants with individual deletions of all two-component systems and several related genes, including creBC-regulated genes (cbrA and cbrBC), phoBR-regulated genes (phoA, phoH, phnCDEFGHIJKLMNOP, psiE, and ugpBAECQ), csgD, luxS, and rpoS. The results of this battery of nearly 200,000 tests provided a wealth of new information concerning many of these systems. Of 37 different two-component mutants, 22 showed altered phenotypes. Many phenotypes were expected, and several new phenotypes were also revealed. The results are discussed in terms of the biological roles and other information concerning these systems, including DNA microarray data for a large number of the same mutants. Other mutational effects are also discussed.     

Rewiring the specificity of two-component signal transduction systems

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.