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.     

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.     

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.     

Mutational analysis of repression and activation of the tyrP gene in Escherichia coli.

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.     

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.     

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.     

The torYZ (yecK bisZ) operon encodes a third respiratory trimethylamine N-oxide reductase in Escherichia coli

The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii) torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The torYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, no torD homologue is found in the sequences around torYZ. Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide.