Jacques Covès

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.

Ferric reductases or flavin reductases

Assimilation of iron by microorganisms requires the presence of ferric reductases which participate in the mobilization of iron from ferrisiderophores. The common structural and catalytic properties of these enzymes are described and shown to be identical to those of flavin reductases. This strongly suggests that, in general, the reduction of iron depends on reduced flavins provided by flavin reductases.

Mobilization of selenite by Ralstonia metallidurans CH34.

ABSTRACT Ralstonia metallidurans CH34 (formerlyAlcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metalliduransCH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.

Solubilization and partial purification of UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope

We have developed procedures to solubilize the envelope UDP‐galactose:diacylglycerol galactosyltransferase activity and to assay this enzyme after solubilization with a zwitterionic non‐denaturing detergent (CHAPS) and fractionation. From solubilized envelope membranes isolated from intact spinach chloroplasts, we were able to prepare by chromatography on hydroxyapatite a fraction enriched (6–7‐fold) in this enzyme responsible for monogalactosyldiacylglycerol synthesis.

Optimized set of two-dimensional experiments for fast sequential assignment, secondary structure determination, and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional 1H-15N HSQC-like spectra. The 1H-15N correlation peaks are frequency shifted by an amount ± ωX along the 15N dimension, where ωX is the Cα, Cβ, or Hα frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.

Adaptation of the Wine Bacterium Oenococcus oeni to Ethanol Stress: Role of the Small Heat Shock Protein Lo18 in Membrane Integrity

ABSTRACT Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

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.

Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase?

In MerT, the mercury transporter, a first cysteine pair, located in the first trans‐membrane helix, receives mercury from the periplasm. Then, a second cysteine pair, housed in a cytoplasmic loop connecting the second and the third trans‐membrane helices, is thought to transfer the metal to another cysteine pair located in the N‐terminal extension of the mercuric reductase. We found that a 23‐amino acid synthetic peptide corresponding to the cytoplasmic loop can bind one mercury atom per molecule and that this mercury atom can be transferred specifically to MerAa. The solution structure of Hg‐bound ppMerT has been solved by 1H NMR spectroscopy.

NADPH-sulfite reductase flavoprotein from Escherichia coli: contribution to the flavin content and subunit interaction.

The flavoprotein component (SiR‐FP) of the sulfite reductase of E. coli is an octamer of the 66 kDa α subunit. It was shown to be cleaved in two peptide fragments. The 23 kDa fragment has been purified as a polymer of 8–10 subunits. It corresponds to the N‐terminal part of the native protein and was shown to contain essentially FMN as cofactor. The 43 kDa fragment is monomeric. It contains exclusively FAD and remains able to catalyze efficiently NADPH‐dependent reductions. One can conclude that each α‐chain of SiR‐FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN‐binding domains cooperate for a head‐to‐head subunit interaction.

Structural Basis for Metal Sensing by Cnrx.

CnrX is the metal sensor and signal modulator of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34 that is involved in the setup of cobalt and nickel resistance. We have determined the atomic structure of the soluble domain of CnrX in its Ni-bound, Co-bound, or Zn-bound form. Ni and Co ions elicit a biological response, while the Zn-bound form is inactive. The structures presented here reveal the topology of intraprotomer and interprotomer interactions and the ability of metal-binding sites to fine-tune the packing of CnrX dimer as a function of the bound metal. These data suggest an allosteric mechanism to explain how the complex is switched on and how the signal is modulated by Ni or Co binding. These results provide clues to propose a model for signal propagation through the membrane in the complex.