The bacterial thiopurine methyltransferase tellurite resistance process is highly dependent upon aggregation properties and oxidative stress response.

Bacterial thiopurine methyltransferases (bTPMTs) can favour resistance towards toxic tellurite oxyanions through a pathway leading to the emission of a garlic-like smell. Gene expression profiling completed by genetic, physiological and electron microscopy analyses was performed to identify key bacterial activities contributing to this resistance process. Escherichia coli strain MG1655 expressing the bTPMT was used as a cell model in these experiments. This strain produced a garlic-like smell which was found to be due to dimethyl telluride, and cell aggregates in culture media supplemented with tellurite. Properties involved in aggregation were correlated with cell attachment to polystyrene, which increased with tellurite concentrations. Gene expression profiling supported a role of adhesins in the resistance process with 14% of the tellurite-regulated genes involved in cell envelope, flagella and fimbriae biogenesis. Other tellurite-regulated genes were, at 27%, involved in energy, carbohydrate and amino acid metabolism including the synthesis of antioxidant proteins, and at 12% in the synthesis of transcriptional regulators and signal transduction systems. Escherichia coli mutants impaired in tellurite-regulated genes showed ubiquinone and adhesins synthesis, oxidative stress response, and efflux to be essential in the bTPMT resistance process. High tellurite resistance required a synergistic expression of these functions and an efficient tellurium volatilization by the bTPMT.

Characterization of the MerD protein from Ralstonia metallidurans CH34: a possible role in bacterial mercury resistance by switching off the induction of the mer operon.

MerD and MerR from Tn4378 found in Ralstonia metallidurans CH34 were purified to homogeneity after overexpression in Escherichia coli. Using electrophoretic mobility shift assays and footprinting experiments, we found that MerD cannot bind to DNA. However, in vitro MerD can form a ternary complex in association with merOP and MerR. The presence of MerD in this complex was demonstrated by Western analysis with antibodies to MerD. To our knowledge, this is the first description of such a ternary complex between MerD-MerR and DNA. The formation and stability of this ternary complex are dependent on the relative concentration of the two proteins and modulated by the presence of mercury. We postulate that MerD could displace Hg-bound MerR from the mer operator to allow new synthesis of metal-free MerR able to switch off the induction of the mer genes when the external mercury is exhausted. This could fully explain how MerD can be a co-regulator repressing the induction of the mer operon.

Biophysical characterization of the MerP-like amino-terminal extension of the mercuric reductase from Ralstonia metallidurans CH34.

The purified native mercuric reductase (MerA) from Ralstonia metallidurans CH34 contains an N-terminal sequence of 68 amino acids predicted to be homologous to MerP, the periplasmic mercury-binding protein. This MerP-like protein has now been expressed independently. The protein was named MerAa by homology with Ccc2a, the first soluble domain of the copper-transporting ATPase from yeast. Deltaa has been characterized using a set of biophysical techniques. The binding of mercury was followed using circular dichroism spectroscopy and electrospray mass spectrometry. The two cysteine residues contained in the consensus sequence GMTC XXC are involved in the binding of one mercury atom, with an apparent affinity comparable to that of MerP for the same metal. The metal-binding site is confirmed by NMR chemical shift changes observed between apo- and metal-bound MerAa in solution. NMR shift and NOE data also indicate that only minor structural changes occur upon metal binding. Further NMR investigation of the fold of MerAa using long-range methyl-methyl NOE and backbone residual dipolar coupling data confirm the expected close structural homology with MerP. (15)N relaxation data show that MerAa is a globally rigid molecule. An increased backbone mobility was observed for the loop region connecting the first beta-strand and the first alpha-helix and comprising the metal-binding domain. Although significantly reduced, this loop region keeps some conformational flexibility upon metal binding. Altogether, our data suggest a role of MerAa in mercury trafficking.

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain.

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.