An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO

The osmoregulator trimethylamine-N-oxide (TMAO), commonplace in aquatic organisms, is used as the terminal electron acceptor for respiration in many bacterial species. The TMAO reductase (Tor) pathway for respiratory catalysis is controlled by a receptor system that comprises the TMAO-binding protein TorT, the sensor histidine kinase TorS, and the response regulator TorR. Here we study the TorS/TorT sensor system to gain mechanistic insight into signaling by histidine kinase receptors. We determined crystal structures for complexes of TorS sensor domains with apo TorT and with TorT (TMAO); we characterized TorS sensor associations with TorT in solution; we analyzed the thermodynamics of TMAO binding to TorT-TorS complexes; and we analyzed in vivo responses to TMAO through the TorT/TorS/TorR system to test structure-inspired hypotheses. TorS-TorT(apo) is an asymmetric 2:2 complex that binds TMAO with negative cooperativity to form a symmetric active kinase.     

TorC apocytochrome negatively autoregulates the trimethylamine N-oxide (TMAO) reductase operon in Escherichia coli

The trimethylamine N-oxide (TMAO) anaerobic respiratory system of Escherichia coli comprises a periplasmic terminal TMAO reductase (TorA) and a pentahaem c-type cytochrome (TorC), which is involved in electron transfer to TorA. The structural proteins are encoded by the torCAD operon whose expression is induced in the presence of TMAO through the TorS/TorR two-component system. By using a genomic library cloned into a multicopy plasmid, we identified TorC as a possible negative regulator of the tor operon. Interestingly, in trans overexpression of torC not only decreased the activity of a torA'-'lacZ fusion, but also dramatically reduced the amount of mature TorC cytochrome. This led us to propose that, after translocation, TorC apocytochrome downregulates the tor operon unless it is properly matured. In agreement with this hypothesis, we have shown that mini-Tn10 insertions within genes involved in the c-type cytochrome maturation pathway or haem biosynthesis decreased tor operon expression. Dithiothreitol (DTT), which reduces disulphide bonds and thus prevents the first step in c-type cytochrome formation, also strongly decreases the tor promoter activity. The DTT effect is TorC dependent, as it is abolished when torC is disrupted. In contrast, overexpression of the c-type cytochrome maturation (ccm ) genes relieved the tor operon of the negative control and allowed the bacteria to produce a higher amount of TorC holocytochrome. Therefore, the TorC negative autoregulation probably means that maturation of the c-type cytochrome is a limiting step for Tor system biogenesis. Genetic experiments have provided evidence that TorC control is mediated by the TorS/TorR two-component system and different from the tor anaerobic control. In our working model, TMAO and apoTorC bind to the periplasmic side of TorS, but TMAO activates TorS autophosphorylation, whereas apoTorC inhibits the TorS kinase activity.     

An unorthodox sensor protein (TorS) mediates the induction of the tor structural genes in response to trimethylamine N-oxide in Escherichia coli

We isolated and characterized three spontaneous mutations leading to trimethylamine N-oxide (TMAO)-independent expression of the tor operon encoding the TMAO-reductase anaerobic respiratory system in Escherichia coli. The mutations lie in a new tor regulatory gene, the torS gene, which probably encodes a sensor protein of a two-component regulatory system. One mutation, which leads to full TMAO-constitutive expression, is a 3-amino-acid deletion within the potential N-terminal periplasmic region, suggesting that this region contains the TMAO-detector site. For the other two mutations, a further induction of the tor operon is observed when TMAO is added. Both are single substitutions and affect the linker region located between the detector and the conserved transmitter domains. Thus, as proposed for other sensors, the TorS linker region might play an essential role in propagating conformational changes between the detector and the cytoplasmic signalling regions. The TorR histidine kinase is an unorthodox sensor that contains a receiver and a C-terminal alternative transmitter domain in addition to the domains found in most sensors. Previously, we showed that TMAO induction of the tor operon requires the TorR response regulator and the TorT periplasmic protein. Additional genetic data confirm that torS encodes the sensor partner of TorR and TorT. First, insertion within torS abolishes tor operon expression whatever the growth conditions. Second, overexpressed TorR bypasses the requirement for torS, whereas the torT gene product is dispensable for tor operon expression in a torS constitutive mutant. This supports a signal-transduction cascade from TorT to TorR via TorS.     

TorT, a member of a new periplasmic binding protein family, triggers induction of the Tor respiratory system upon trimethylamine N-oxide electron-acceptor binding in Escherichia coli

In anaerobiosis, Escherichia coli can use trimethylamine N-oxide (TMAO) as a terminal electron acceptor. Reduction of TMAO in trimethylamine (TMA) is mainly performed by the respiratory TMAO reductase. This system is encoded by the torCAD operon, which is induced in the presence of TMAO. This regulation involves a two-component system comprising TorS, an unorthodox histidine kinase, and TorR, a response regulator. A third protein, TorT, sharing homologies with periplasmic binding proteins, plays a key role in this regulation because disruption of the torT gene abolishes tor expression. In this study we showed that TMAO protects TorT against degradation by the GluC endoproteinase and modifies its temperature-induced CD spectrum. We also isolated a TorT negative mutant that is no longer protected by TMAO from degradation by GluC. Isothermal titration calorimetry confirmed that TorT binds TMAO with a binding constant of 150 mum. Therefore, we conclude that TorT binds TMAO and that this binding promotes a conformational change of TorT. We also showed that TorT interacts with the periplasmic domain of TorS in both the presence and absence of TMAO but the TorT-TMAO complex induces a higher GluC protection of TorS than TorT alone. These results support the idea that TMAO binding to TorT induces a cascade of conformational changes from TorT to TorS, leading to TorS activation. We identified several homologues of the TorT protein that define a new family of periplasmic binding proteins. We thus propose that the members of this family bind TMAO or related compounds and that they are involved in signal transduction or even substrate transport.     

Effects of ISSo2 insertions in structural and regulatory genes of the trimethylamine oxide reductase of Shewanella oneidensis

We have isolated three Shewanella oneidensis mutants specifically impaired in trimethylamine oxide (TMAO) respiration. The mutations arose from insertions of an ISSo2 element into torA, torR, and torS, encoding, respectively, the TMAO reductase TorA, the response regulator TorR, and the sensor TorS. Although TorA is not the sole enzyme reducing TMAO in S. oneidensis, growth analysis showed that it is the main respiratory TMAO reductase. Use of a plasmid-borne torE'-lacZ fusion confirmed that the TorS-TorR phosphorelay mediates TMAO induction of the torECAD operon.     

大肠杆菌TorS/TorR二组分体应答蛋白TorR对DNA复制起始的影响

二组分体作为一种信号转导系统在细菌中普遍存在,能够感知外界环境变化并做出应答。细菌中CckA/CtrA、ArcA/ArcB和PhoP/PhoQ二组分体与DNA复制起始和细胞分裂相关,但目前还未见TorS/TorR二组分体对细胞周期及DNA复制影响的相关报道。大肠杆菌TorS/TorR二组分体能够监测细胞周围氧化三甲胺(Trimethylamine oxide, TMAO)的浓度变化,但其是否影响DNA复制起始呢?文章利用流式细胞仪检测了ΔtorS和ΔtorR突变体菌株的复制式样。结果发现,ΔtorS突变菌株每个细胞复制起始原点数目和倍增时间与野生型细胞一致,而ΔtorR突变菌株每个细胞复制起始原点数目多于野生型细胞,说明复制起始发生时间比野生型细胞早。但是过表达TorR蛋白或者共同表达TorS和TorR蛋白都不能使ΔtorR突变体表型恢复为野生型表型。而在野生型和ΔtorR突变细胞中过表达SufD蛋白能使复制起始提早发生,在ΔtorR和ΔsufD双突变细胞中复制起始延迟。所以,TorR可能通过改变sufD基因的表达来间接影响染色体复制起始。     

A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC --> YojN --> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour

In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --> RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His --> Asp phosphorelay, this RcsC --> RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His --> Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC --> YojN --> RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.     

Amplification of signaling activity of the arc two-component system of Escherichia coli by anaerobic metabolites. An in vitro study with different protein modules

In Escherichia coli, changes in redox condition of growth are sensed and signaled by the Arc two-component system. This system consists of ArcB as the membrane-associated sensor kinase and ArcA as the cytoplasmic response regulator. ArcB is a tripartite kinase, possessing a primary transmitter, a receiver, and a secondary transmitter domain that catalyzes the phosphorylation of ArcA via a His --> Asp --> His --> Asp phosphorelay, as well as the dephosphorylation of ArcA-P by a reverse phosphorelay. When ArcA and ArcB were incubated with ATP, the peak levels of phosphorylated proteins increased in the presence of the fermentation metabolites D-lactate, acetate, or pyruvate. In this study, we report that these effectors accelerate the autophosphorylation activity of ArcB and enhance the transphosphorylation of ArcA, but have no effect on the dephosphorylation of ArcA-P. Moreover, the presence of the receiver domain of ArcB is essential for the effectors to influence the autophosphorylation rate of the primary transmitter domain of ArcB.     

Binding of the TorR regulator to cis-acting direct repeats activates tor operon expression

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N-oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC'-lacZ fusion dramatically reduces TorR-dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel-retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the −35 promoter sequence and includes the third tor box (box3); the last is found downstream from the −35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments.
We propose a model in which multiple binding sites (i.e. the tor boxes) contribute to the formation of a nucleoprotein complex, but only one particular proximal site positions TorR properly so that it interacts with RNA polymerase.     

The Sensory Histidine Kinases TorS and EvgS Tend to Form Clusters in Escherichia coli Cells

Microorganisms use multiple two-component sensory systems to detect changes in their environment and elicit physiological responses. Despite their wide spread and importance, the intracellular organization of two-component sensory proteins in bacteria remains little investigated. A notable exception is the well-studied clustering of the chemoreceptor-kinase complexes that mediate chemotaxis behaviour. However, these chemosensory complexes differ fundamentally from other systems, both structurally and functionally. Therefore, studying the organization of typical sensory kinases in bacteria is essential for understanding the general role of receptor clustering in bacterial sensory signalling. Here, by studying mYFP-tagged sensory kinases in Escherichia coli, we show that the tagged TorS and EvgS sensors have a clear tendency for self-association and clustering. These sensors clustered even when expressed at a level of a few hundred copies per cell. Moreover, the mYFP-tagged response regulator TorR showed clear TorS-dependent clustering, indicating that untagged TorS sensors also tend to form clusters. We also provide evidence for the functionality of these tagged sensors. Experiments with truncated TorS or EvgS proteins suggested that clustering of EvgS sensors depends on the cytoplasmic part of the protein, whereas clustering of TorS sensors can be potentially mediated by the periplasmic/transmembrane domain. Overall, these findings support the notion that sensor clustering plays a role in bacterial sensory signalling beyond chemotaxis.     

Compilation and characterization of histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in plants: AHP signal transducers of Arabidopsis thaliana

Histidine (His)-to-Aspartate (Asp) phosphorelay signal transduction systems are generally made up of a "sensor histidine (His)-kinase", a "response regulator", and a "histidine-containing phosphotransmitter (HPt)". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that the His-to-Asp phosphorelay mechanism is at least partly responsible for propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we compiled the members of the HPt family of phosphotransmitters in Arabidopsis thaliana (AHP-series, Arabidopsis HPt phosphotransmitters), based on both database and experimental analyses, in order to provide a comprehensive basis at the molecular level for understanding the function of the AHP phosphotransmitters that are implicated in the His-to-Asp phosphorelay of higher plants.     

Compilation and Characterization of Histidine-Containing Phosphotransmitters Implicated in His-to-Asp Phosphorelay in Plants: AHP Signal Transducers of Arabidopsis thaliana

Histidine (His)-to-Aspartate (Asp) phosphorelay signal transduction systems are generally made up of a “sensor histidine (His)-kinase”, a “response regulator”, and a “histidine-containing phosphotransmitter (HPt)”. In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that the His-to-Asp phosphorelay mechanism is at least partly responsible for propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we compiled the members of the HPt family of phosphotransmitters in Arabidopsis thaliana (AHP- series, Arabidopsis HPt phosphotransmitters), based on both database and experimental analyses, in order to provide a comprehensive basis at the molecular level for understanding the function of the AHP phosphotransmitters that are implicated in the His-to-Asp phosphorelay of higher plants.