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.     

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.     

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.     

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。     

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.     

The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli.

Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway.     

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 histidine kinase CusS senses silver ions through direct binding by its sensor domain

The Cus system of Escherichia coli aids in protection of cells from high concentrations of Ag(I) and Cu(I). The histidine kinase CusS of the CusRS two-component system functions as a Ag(I)/Cu(I)-responsive sensor kinase and is essential for induction of the genes encoding the CusCFBA efflux pump. In this study, we have examined the molecular features of the sensor domain of CusS in order to understand how a metal-responsive histidine kinase senses specific metal ions. We find that the predicted periplasmic sensor domain of CusS directly interacts with Ag(I) ions and undergoes a conformational change upon metal binding. Metal binding also enhances the tendency of the domain to dimerize. These findings suggest a model for activation of the histidine kinase through metal binding events in the periplasmic sensor domain.     

Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagic Escherichia coli (EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression of torR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression of glpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.     

Interdomain interactions within the two-component heme-based sensor DevS from Mycobacterium tuberculosis

DevS is the sensor of the DevS-DevR two-component regulatory system of Mycobacterium tuberculosis. This system is thought to be responsible for initiating entrance of this bacterium into the nonreplicating persistent state in response to NO and anaerobiosis. DevS is modular in nature and consists of two N-terminal GAF domains and C-terminal histidine kinase and ATPase domains. The first GAF domain (GAF A) binds heme, and this cofactor is thought to be responsible for sensing environmental stimuli, but the function of the second GAF domain (GAF B) is unknown. Here we report the RR characterization of full-length DevS (FL DevS) as well as truncated proteins consisting of the single GAF A domain (GAF A DevS) and both GAF domains (GAF A/B) in both oxidation states and bound to the exogenous ligands CO, NO, and O2. The results indicate that the GAF B domain increases the specificity with which the distal heme pocket of the GAF A domain interacts with CO and NO as opposed to O2. Specifically, while two comparable populations of CO and NO adducts are observed in GAF A DevS, only one of these two conformers is present in significant concentration in the GAF A/B and FL DevS proteins. In contrast, hydrogen bond interactions at the bound oxygen in the oxy complexes are conserved in all DevS constructs. The comparison of the data obtained with the O2 complexes with those of the CO and NO complexes suggests a model for ligand discrimination which relies on a specific hydrogen-bonding network with bound O2. It also suggests that interactions between the two GAF domains are responsible for transduction of structural changes at the heme domain that accompany ligand binding/dissociation to modulate activity at the kinase domain.