Three (and more) component regulatory systems – auxiliary regulators of bacterial histidine kinases

Two‐component signal transduction (TCST) is the most prevalent mechanism employed by microbes to sense and respond to environmental changes. It is characterized by the signal‐induced transfer of phosphate from a sensor histidine kinase (HK) to a response regulator (RR), resulting in a cellular response. An emerging theme in the field of TCST signalling is the discovery of auxiliary factors, distinct from the HK and RR, which are capable of influencing phosphotransfer. One group of TCST auxiliary proteins accomplishes this task by acting on HKs. Auxiliary regulators of HKs are widespread and have been identified in all cellular compartments, where they can influence HK activity through interactions with the sensing, transmembrane or enzymatic domains of the HK. The effects of an auxiliary regulator are controlled by its regulated expression, modification and/or through ligand binding. Ultimately, auxiliary regulators can connect a given TCST system to other regulatory networks in the cell or result in regulation of the TCST system in response to an expanded range of stimuli. The studies highlighted in this review draw attention to an emerging view of bacterial TCST systems as core signalling units upon which auxiliary factors act.     

Bacterial 2-component signal transduction systems: a fluorescence polarization screen for response regulator-protein binding

Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the Van RSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements.     

Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

Evolution of two-component signal transduction

Two-component signal transduction (TCST) systems are the principal means for coordinating responses to environmental changes in bacteria as well as some plants, fungi, protozoa, and archaea. These systems typically consist of a receptor histidine kinase, which reacts to an extracellular signal by phosphorylating a cytoplasmic response regulator, causing a change in cellular behavior. Although several model systems, including sporulation and chemotaxis, have been extensively studied, the evolutionary relationships between specific TCST systems are not well understood, and the ancestry of the signal transduction components is unclear. Phylogenetic trees of TCST components from 14 complete and 6 partial genomes, containing 183 histidine kinases and 220 response regulators, were constructed using distance methods. The trees showed extensive congruence in the positions of 11 recognizable phylogenetic clusters. Eukaryotic sequences were found almost exclusively in one cluster, which also showed the greatest extent of domain variability in its component proteins, and archaeal sequences mainly formed species-specific clusters. Three clusters in different parts of the kinase tree contained proteins with serine-phosphorylating activity. All kinases were found to be monophyletic with respect to other members of their superfamily, such as type II topoisomerases and Hsp90. Structural analysis further revealed significant similarity to the ATP-binding domain of eukaryotic protein kinases. TCST systems are of bacterial origin and radiated into archaea and eukaryotes by lateral gene transfer. Their components show extensive coevolution, suggesting that recombination has not been a major factor in their differentiation. Although histidine kinase activity is prevalent, serine kinases have evolved multiple times independently within this family, accompanied by a loss of the cognate response regulator(s). The structural and functional similarity between TCST kinases and eukaryotic protein kinases raises the possibility of a distant evolutionary relationship.     

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。     

Bacterial histidine kinase as signal sensor and transducer

Adaptation to an environmental stress is essential for cell survival in all organisms, from E. coli to human. To respond to changes in their surroundings, bacteria utilize two-component systems (TCSs), also known as histidyl-aspartyl phosphorelay (HAP) systems that consist of a histidine kinase (HK) sensor and a cognate response regulator (RR). While mammals developed complex signaling systems involving serine/threonine/tyrosine kinases in stress response mechanisms, bacterial TCS/HAP systems represent a simple but elegant prototype of signal transduction machineries. HKs are known as a seductive target for anti-bacterial therapeutic development, because of their significance in pathological virulence in some bacteria such as Salmonella enterica. Recent molecular and structural studies have shed light on the molecular basis of the signaling mechanism of HK sensor kinases. This review will focus on recent advancements in structural investigation of signal sensing and transducing mechanisms by HKs, which is critical to our understanding of bacterial biology and pathology.     

The HWE histidine kinases, a new family of bacterial two-component sensor kinases with potentially diverse roles in environmental signaling

Two-component signal transduction pathways play a major role in the response of bacteria to external cues. These pathways are initiated by large collection of histidine kinases (HKs) containing a sensor domain that perceives the environmental signal followed by an HK domain that triggers a histidine-aspartate phosphorelay. Previous phylogenetic analyses identified 11 major families of two-component HKs by comparing signature motifs within the HK domain. Here we describe a new family with homology to Agrobacterium tumefaciens BphP2, an HK first discovered by the presence of a phytochrome sensor domain involved in light perception. Members of this sensor HK family differ from most others by the absence of a recognizable F box and the presence of several uniquely conserved residues, including a histidine in the N box and a tryptophan-X-glutamic acid sequence in the G1 box, which we have used to define the family (HWE). At least 81 members were identified in a variety of alpha- and gamma-proteobacteria, with a significant enrichment in the Rhizobiaceae family. Several representatives were shown to have HK activity in vitro, supporting their proposed participation in phosphorelays. One or more domains related to signal transduction were evident N-terminal to the HK domain, including chemotactic methyltransferase domains, suggesting that this family has multiple roles in environmental signaling. The discovery of the HWE family further extends the diversity within the HK superfamily and expands the importance of two-component signaling in bacteria.     

Two-Component Signal Transduction Systems, Environmental Signals, and Virulence

The relevance toward virulence of a variety of two-component signal transduction systems is reviewed for 16 pathogenic bacteria, together with the wide array of environmental signals or conditions that have been implicated in their regulation. A series of issues is raised, concerning the need to understand the environmental cues that determine their regulation in the infected host and in the environment outside the laboratory, which shall contribute toward the bridging of bacterial pathogenesis and microbial ecology.     

Comparative Genomic and Phylogenetic Analyses Reveal the Evolution of the Core Two-Component Signal Transduction Systems in Enterobacteria

The two-component signal transduction system (TCST) consists of a histidine kinase (HK) and a response regulator (RR). TCSTs play important roles in sensing and reacting to environmental changes, and in bacterial pathogenesis. Previously, we have identified and characterized TCSTs in Erwinia amylovora, a severe plant enterobacterial pathogen, at genome-wide level. Here we conducted a comparative genomic analysis of TCSTs in 53 genomes of 16 enterobacterial species. These species include important plant, animal, human, and insect pathogenic, saprophytic or symbiotic microorganisms. Comparative genomic analysis revealed that enterobacteria contain eight pairs of core TCSTs. Phylogenetic trees reconstructed from a concatenation of the core set of TCSTs from enterobacteria and for individual TCST proteins from species in Proteobacteria showed that most TCST protein trees in the Enterobacteriaceae or in species of the γ-Proteobacteria agreed well with that of the corresponding 16S rRNA gene. It also showed that co-evolutionary relationships existed between cognate partners of the HKs and RRs. Several core TCSTs were quite ancient and universal based on phylogenomic analysis of protein structures. These results indicate that the core TCSTs are relatively conserved, and suggest that these enterobacteria may have maintained their ancient core TCSTs and might acquire specific new TCSTs for their survival in different environments or hosts, or may have evolved new functionalities of the core TCSTs for adaptation to different ecological niches.     

The mechanism of signal transduction by two-component systems

Two-component systems, composed of a homodimeric histidine kinase (HK) and a response regulator (RR), are major signal transduction devices in bacteria. Typically the signal triggers HK autophosphorylation at one His residue, followed by phosphoryl transfer from the phospho-His to an Asp residue in the RR. Signal extinction frequently involves phospho-RR dephosphorylation by a phosphatase activity of the HK. Our understanding of these reactions and of the determinants of partner specificity among HK-RR couples has been greatly increased by recent crystal structures and biochemical experiments on HK-RR complexes. Cis-autophosphorylation (one subunit phosphorylates itself) occurs in some HKs while trans-autophosphorylation takes place in others. We review and integrate this new information, discuss the mechanism of the three reactions and propose a model for transmembrane signaling by these systems.     

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.     

One-component systems dominate signal transduction in prokaryotes

Two-component systems that link environmental signals to cellular responses are viewed as the primary mode of signal transduction in prokaryotes. By analyzing information encoded by 145 prokaryotic genomes, we found that the majority of signal transduction systems consist of a single protein that contains input and output domains but lacks phosphotransfer domains typical of two-component systems. One-component systems are evolutionarily older, more widely distributed among bacteria and archaea, and display a greater diversity of domains than two-component systems.     

Histidine kinases and response regulators in networks

Two-component systems, composed of a histidine kinase (HK) and a response regulator (RR), are the major signal transduction devices in bacteria. Originally it was thought that these two components function as linear, phosphorylation-driven stimulus-response system. Here, we will review how accessory proteins are employed by HKs and RRs to mediate signal integration, scaffolding, interconnection and allosteric regulation, and how these two components are embedded in regulatory networks.     

Mutational analysis of dimeric linkers in peri- and cytoplasmic domains of histidine kinase DctB reveals their functional roles in signal transduction

Membrane-associated histidine kinases (HKs) in two-component systems respond to environmental stimuli by autophosphorylation and phospho-transfer. HK typically contains a periplasmic sensor domain that regulates the cytoplasmic kinase domain through a conserved cytoplasmic linker. How signal is transduced from the ligand-binding site across the membrane barrier remains unclear. Here, we analyse two linker regions of a typical HK, DctB. One region connects the first transmembrane helix with the periplasmic Per-ARNT-Sim domains, while the other one connects the second transmembrane helix with the cytoplasmic kinase domains. We identify a leucine residue in the first linker region to be essential for the signal transduction and for maintaining the delicate balance of the dimeric interface, which is key to its activities. We also show that the other linker, belonging to the S-helix coiled-coil family, plays essential roles in signal transduction inside the cell. Furthermore, by combining mutations with opposing activities in the two regions, we show that these two signalling transduction elements are integrated to produce a combined effect on the final activity of DctB.     

Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of the most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.     

Evolution of prokaryotic two-component system signaling pathways: gene fusions and fissions

Two-component systems (TCSs) are common signal transduction systems, typically comprising paired histidine protein kinase (HK) and response regulator (RR) proteins. In many examples, it appears RR and HK genes have fused, producing a "hybrid kinase " We have characterized a set of prokaryotic genes encoding RRs, HKs, and hybrid kinases, enabling characterization of gene fusion and fission. Primary factors correlating with fusion rates are the presence of transmembrane helices in HKs and the presence of DNA-binding domains in RRs, features that require correct (and separate) spatial location. In the absence of such features, there is a relative abundance of fused genes. The order of paired HK and RR genes and the nucleotide distance between encoded domains also correlate with apparent gene fusion rates. We propose that localization requirements and relative positioning of encoded domains within TCS genes affect the function (and therefore retention) of hybrid kinases resulting from gene fusion.     

Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria.     

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to “sense” environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is “sensing” disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are “sensed” by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.     

Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5'-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.