Proteome analysis of outer membrane vesicles from a clinical Acinetobacter baumannii isolate

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor , 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor . Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor .     

Functional insights from the molecular modelling of a novel two-component system

Two-component systems (TCSs) are the major signalling pathway in bacteria and represent potential drug targets. Among the 11 paired TCS proteins present in Mycobacterium tuberculosis H37Rv, the histidine kinases (HKs) Rv0600c (HK1) and Rv0601c (HK2) are annotated to phosphorylate one response regulator (RR) Rv0602c (TcrA). We wanted to establish the sequence-structure-function relationship to elucidate the mechanism of phosphotransfer using in silico methods. Sequence alignments and codon usage analysis showed that the two domains encoded by a single gene in homologous HKs have been separated into individual open-reading frames in M. tuberculosis. This is the first example where two incomplete HKs are involved in phosphorylating a single RR. The model shows that HK2 is a unique histidine phosphotransfer (HPt)-mono-domain protein, not found as lone protein in other bacteria. The secondary structure of HKs was confirmed using "far-UV" circular dichroism study of purified proteins. We propose that HK1 phosphorylates HK2 at the conserved H131 and the phosphoryl group is then transferred to D73 of TcrA.     

Two-component signal transduction systems of Xanthomonas spp.: a lesson from genomics

The two-component signal transduction systems (TCSTSs), consisting of a histidine kinase sensor (HK) and a response regulator (RR), are the dominant molecular mechanisms by which prokaryotes sense and respond to environmental stimuli. Genomes of Xanthomonas generally contain a large repertoire of TCSTS genes (approximately 92 to 121 for each genome), which encode diverse structural groups of HKs and RRs. Among them, although a core set of 70 TCSTS genes (about two-thirds in total) which accumulates point mutations with a slow rate are shared by these genomes, the other genes, especially hybrid HKs, experienced extensive genetic recombination, including genomic rearrangement, gene duplication, addition or deletion, and fusion or fission. The recombinations potentially promote the efficiency and complexity of TCSTSs in regulating gene expression. In addition, our analysis suggests that a co-evolutionary model, rather than a selfish operon model, is the major mechanism for the maintenance and microevolution of TCSTS genes in the genomes of Xanthomonas. Genomic annotation, secondary protein structure prediction, and comparative genomic analyses of TCSTS genes reviewed here provide insights into our understanding of signal networks in these important phytopathogenic bacteria.     

Dual-histidine kinases in basidiomycete fungi

Dual-histidine kinases (HKs) are complex hybrid HKs containing in a single polypeptide two HK transmitter modules (T) and two-response regulator received domains (R) that are combined in a TRTR geometry. In fungi, this protein family is limited to some particular species of the phylum Basidiomycota and absent in the other phyla. This study extends the investigation of dual-HKs to 80 fully sequenced genomes of basidiomycetes, analyzing their distribution, domain architecture and phylogenetic relationships. Moreover, similarly to dual-HKs of basidiomycetes, several species of bacteria were found that contain hybrid HKs with a TRTR domain architecture encoded in a single gene.     

Building interacting partner predictors using co-varying residue pairs between histidine kinase and response regulator pairs of 48 bacterial two-component systems

The two‐component system (TCS) is a signal transduction system that involves a histidine kinase (HK) and a response regulator (RR). Although up to hundreds of TCSs may operate in parallel in a bacterial cell, the high‐fidelity of a TCS signaling is well maintained, minimizing irrelevant crosstalk between TCSs. When a HK gene and a RR gene in a given TCS system exist in neighboring positions, it is almost certain that their protein products (i.e., HK and RR) are interacting partners. However, large bacterial genomes often have multiple HK genes and/or cognate RR genes that are not neighboring positions. In many partially assembled genomes, some HK genes and RR genes belong to different contigs. In these cases, it is not clear which HK(s) and RR(s) interact. By combining information‐theoretic and graph‐theoretic approaches, we developed a computational method identifying co‐evolving residue pairs between HKs and cognate RRs and predicting the interacting HK:RR pairs for each TCS. In addition, we built a TCSppWWW webserver ( http://compath.org/platcom/tcs ) that takes query sequences of pairing candidates and predicts their HK:RR pairing using precomputed models. The current release of TCSppWWW provides predictors for 48 TCSs using over 20,000 protein sequences from about 900 bacterial genomes. Three different types of predictors using Random Forest, RBF Network, and Naïve Bayes are provided. Once a set of HK and RR candidate sequences are submitted, TCSppWWW aligns query sequences to the precomputed multiple sequence alignment of HK:RR pairs, extracts co‐evolving column positions, then returns prediction results with prediction margin and additional information. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

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.     

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.     

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei

Lactobacillus casei has traditionally been recognized as a probiotic, thus needing to survive the industrial production processes and transit through the gastrointestinal tract before providing benefit to human health. The two-component signal transduction system (TCS) plays important roles in sensing and reacting to environmental changes, which consists of a histidine kinase (HK) and a response regulator (RR). In this study we identified HKs and RRs of six sequenced L. casei strains. Ortholog analysis revealed 15 TCS clusters (HK–RR pairs), one orphan HKs and three orphan RRs, of which 12 TCS clusters were common to all six strains, three were absent in one strain. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. Some TCS clusters are involved with the response under the stress of the bile salts, acid, or oxidative, which contribute to survive the difficult journey through the human gastrointestinal tract. Computational predictions of 15 TCSs were verified by PCR experiments. This genomic level study of TCSs should provide valuable insights into the conservation and divergence of TCS proteins in the L. casei strains.     

Genome-wide comparison of the His-to-Asp phosphorelay signaling components of three symbiotic genera of Rhizobia.

Histidine-to-aspartate (His-Asp) phosphorelay (or two-component) systems are very common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli. The His-Asp phosphorelay components include "sensor histidine kinase (HK)", "phosphotransfer intermediate (HPt)", and "response regulator (RR)". With special reference to three bacterial species (Mesorhizobium loti, Bradyrhizobium japonicum, Sinorhizobium meliloti), each of which belongs to a different genera of Rhizobia, here we attempted to compile all of the His-Asp phosphorelay components in order to reveal a comparative genome-wide overview as to the His-Asp phosphorelay. It was revealed that M. loti has 47 HKs, 1 HPts, and 58 RRs; B. japonicum has 80 HKs, 3 HPts, and 91 RRs; whereas S. meliloti has 40 HKs, 1 HPt, and 58 RRs. These His-Asp phosphorelay components were extensively compiled and characterized. The resulting overview as to the His-Asp phosphorelay of Rhizobia will provide us with a basis for understanding of the fundamental mechanisms underlying interactions between plants and microorganisms (including symbiosis), as well as nitrogen fixation.     

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.     

Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli

Bacteria possess a signal transduction system, referred to as a two-component system, for adaptation to external stimuli. Each two-component system consists of a sensor protein-histidine kinase (HK) and a response regulator (RR), together forming a signal transduction pathway via histidyl-aspartyl phospho-relay. A total of 30 sensor HKs, including as yet uncharacterized putative HKs (BaeS, BasS, CreC, CusS, HydH, RstB, YedV, and YfhK), and a total of 34 RRs, including putative RRs (BaeR, BasR, CreB, CusR, HydG, RstA, YedW, YfhA, YgeK, and YhjB), have been suggested to exist in Escherichia coli. We have purified the carboxyl-terminal catalytic domain of 27 sensor HKs and the full-length protein of all 34 RRs to apparent homogeneity. Self-phosphorylation in vitro was detected for 25 HKs. The rate of self-phosphorylation differed among HKs, whereas the level of phosphorylation was generally co-related with the phosphorylation rate. However, the phosphorylation level was low for ArcB, HydH, NarQ, and NtrB even though the reaction rate was fast, whereas the level was high for the slow phosphorylation species BasS, CheA, and CreC. By using the phosphorylated HKs, we examined trans-phosphorylation in vitro of RRs for all possible combinations. Trans-phosphorylation of presumed cognate RRs by HKs was detected, for the first time, for eight pairs, BaeS-BaeR, BasS-BasR, CreC-CreB, CusS-CusR, HydH-HydG, RstB-RstA, YedV-YedW, and YfhK-YfhA. All trans-phosphorylation took place within less than 1/2 min, but the stability of phosphorylated RRs differed, indicating the involvement of de-phosphorylation control. In addition to the trans-phosphorylation between the cognate pairs, we detected trans-phosphorylation between about 3% of non-cognate HK-RR pairs, raising the possibility that the cross-talk in signal transduction takes place between two-component systems.     

A Non-hydrolyzable ATP Derivative Generates a Stable Complex in a Light-inducible Two-component System

Isothermal calorimetry (ITC) measurements yielded the binding constants during complex formation of light-inducible histidine kinases (HK) and their cognate CheY-type response regulators (RR). HK-RR interactions represent the core function of the bacterial two-component system, which is also present in many bacterial phytochromes. Here, we have studied the recombinant forms of phytochromes CphA and CphB from the cyanobacterium Tolypothrix PCC7601 and their cognate RRs RcpA and RcpB. The interaction between the two reaction partners (HK and RR) was studied in the presence and absence of ATP. A complex formation was observable in the presence of ATP, but specific interactions were only found when a non-hydrolyzable ATP derivative was added to the mixture. Also, the incubation of the HK domain alone (expressed as a recombinant protein) with the RR did not yield specific interactions, indicating that the HK domain is only active as a component of the full-length phytochrome. Considering also previous studies on the same proteins (Hübschmann, T., Jorissen, H. J. M. M., Börner, T., Gärtner, W., and de Marsac, N. (2001) Eur. J. Biochem. 268, 3383–3389) we now conclude that the HK domains of these phytochromes are active only when the chromophore domain is in its Pr form. The formerly documented phosphate transfer between the HK domain and the RR takes place via a transiently formed protein-protein complex, which becomes detectable by ITC in the presence of a non-hydrolyzable ATP derivative. This finding is of interest also in relation to the function of some (blue light-sensitive) photoreceptors that carry the HK domain and the RR fused together in one single protein.     

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.     

单增李斯特菌食品分离株LM201的双组分信号转导系统的生物信息学分析

用生物信息学方法对已完成基因组测序的单增李斯特菌(Listeria monocytogenes,Lm)食品分离株LM201的双组分信号转导系统(Two-component signal transduction systems,TCSs)进行了数量统计、结构分析和功能预测。结果发现:LM201有14对TCSs和2个孤儿应答调控子(Response regulator,RR);其组氨酸激酶(Histidine kinase,HK)具有11种组成结构;其RRs分属于7个亚家族;有3对TCSs和1个孤儿RR的预测功能在Lm中未见报道,有1对TCS功能预测为未知。该研究结果能为构建Lm的TCSs交叉调控网络提供参考,以明确TCSs在Lm毒力调控方面的机制。     

Structure of the entire cytoplasmic portion of a sensor histidine-kinase protein

The large majority of histidine kinases (HKs) are multifunctional enzymes having autokinase, phosphotransfer and phosphatase activities, and most of these are transmembrane sensor proteins. Sensor HKs possess conserved cytoplasmic phosphorylation and ATP-binding kinase domains. The different enzymatic activities require participation by one or both of these domains, implying the need for different conformational states. The catalytic domains are linked to the membrane through a coiled-coil segment that sometimes includes other domains. We describe here the first crystal structure of the complete cytoplasmic region of a sensor HK, one from the thermophile Thermotoga maritima in complex with ADPbetaN at 1.9 A resolution. The structure reveals previously unidentified functions for several conserved residues and reveals the relative disposition of domains in a state seemingly poised for phosphotransfer. The structure thereby inspires hypotheses for the mechanisms of autophosphorylation, phosphotransfer and response-regulator dephosphorylation, and for signal transduction through the coiled-coil segment. Mutational tests support the functional relevance of interdomain contacts.     

Re-engineering the two-component systems as light-regulated in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacteria live in environments with dynamic changes. To sense and respond to different external stimuli, bacteria make use of various sensor-response circuits, called two-component systems (TCSs). A TCS comprises a histidine protein kinase (HK) sensing environmental stimuli and a response regulator protein (RR) regulating downstream genes. The two components are coupled via a phosphorylation control mechanism. In a recent study, we adopted an optogenetics approach to re-engineer the sensor HKs in Escherichia coli as a light-sensing fusion protein. We constructed a light-controllable HK by replacing the original signal-specific sensing domain of HK with the light-sensing domain of Cph1 from Cyanobacteria Synechocystis, so that HK can be investigated by red light. Here, we extended the study to other 16 HK-RR TCSs and constructed a library of light-responsible HK-Cph1 chimeras. By taking the NarX-NarL system as an example, we demonstrated the light responsiveness of the constructed chimera and investigated the frequency response of the NarX-NarL system. The constructed library serves as a toolkit for future TCS study using optogenetics approach.