Diversité des logiques de travail dans les exploitations maraîchères en circuits courts

Les circuits courts alimentaires font aujourd'hui l'objet d'une attention grandissante, aussi bien de la part des producteurs que des consommateurs. Fondés sur une réduction du nombre d'intermédiaires marchands, ces modes de commercialisation répondent à une forte demande de consommation locale et seraient susceptibles de constituer une voie de dynamisation de l'économie agricole locale. Ces systèmes participeraient de plus à un processus de revalorisation économique et sociale du métier d'agriculteur, notamment au travers des liens qu'ils induisent avec les consommateurs et de l'indépendance qu'ils confèrent aux producteurs dans l'exercice de leur activité. Cependant, une des principales limites de ces systèmes de commercialisation serait celle liée à l'organisation du travail et à la gestion du temps sur l'exploitation. Il est en effet souvent souligné que la gestion de la commercialisation combinée à la maitrise d'un système d'exploitation souvent complexe et diversifié entraine une surcharge de travail pour le producteur. Dans cette communication, nous nous intéresserons à la dimension " travail " dans les exploitations maraichères en circuits courts. Nous montrerons que les résultats en termes de temps de travail et de chiffres d'affaire des exploitations semblent dépendre du rapport que les agriculteurs entretiennent avec leur travail, au-delà du fait de commercialiser en circuits courts. Cette notion de rapport au travail sera d'abord vue comme une grille de lecture permettant de mieux cerner la diversité des exploitations maraichères en circuits courts. Elle nous permettra ensuite de mettre en évidence les différentes logiques de travail pouvant expliquer la variabilité des résultats technico-économiques et de soulever les principales problématiques liées à l'organisation du travail dans ces exploitations

Le complexe nitrate réductase A chez Escherichia coli (étude fonctionnelle par spectroscopie RPE et mutagenèse dirigée)

AIX-MARSEILLE1-BU Sci.St Charles (130552104) / SudocSudocFranceF

High-Stability Semiquinone Intermediate in Nitrate Reductase A (NarGHI) from Escherichia coli Is Located in a Quinol Oxidation Site Close to Heme b D †

International audienceThe membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (QD) located in the vicinity of the distal heme bD. Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. 14N and 15N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with 15N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant Aiso(14N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using 14N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the 14N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine Nδ, assigned to the heme bD ligand His-66 residue. Moreover S-band 15N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(15N) = 0.16 MHz). A distance of ∼2.2 Åbetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the QD site of NarGHI

Evidence for an EPR-Detectable Semiquinone Intermediate Stabilized in the Membrane-Bound Subunit NarI of Nitrate Reductase A (NarGHI) from Escherichia coli

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A Robust Genetic System for Producing Heterodimeric Native and Mutant Cytochrome <i>bc</i>₁

The ubihydroquinone:cytochrome <i>c</i> oxidoreductase, or cytochrome <i>bc</i>₁, is central to the production of ATP by oxidative phosphorylation and photophosphorylation in many organisms. Its three-dimensional structure depicts it as a homodimer with each monomer composed of the Fe–S protein, cytochrome <i>b</i>, and cytochrome <i>c</i>₁ subunits. Recent genetic approaches successfully produced heterodimeric variants of this enzyme, providing insights into its mechanism of function. However, these experimental setups are inherently prone to genetic rearrangements as they carry repeated copies of cytochrome <i>bc</i>₁ structural genes. Duplications present on a single replicon (one-plasmid system) or a double replicon (two-plasmid system) could yield heterogeneous populations via homologous recombination or other genetic events at different frequencies, especially under selective growth conditions. In this work, we assessed the origins and frequencies of genetic variations encountered in these systems and describe an improved variant of the two-plasmid system. We found that use of a recombination-deficient background (<i>recA</i>) minimizes spontaneous formation of co-integrant plasmids and renders the homologous recombination within the cytochrome <i>b</i> gene copies inconsequential. On the basis of the data, we conclude that both the newly improved RecA-deficient and the previously used RecA-proficient two-plasmid systems reliably produce native and mutant heterodimeric cytochrome <i>bc</i>₁ variants. The two-plasmid system developed here might contribute to the study of “mitochondrial heteroplasmy”-like heterogeneous states in model bacteria (e.g., <i>Rhodobacter</i> species) suitable for bioenergetics studies. In the following paper (DOI 10.1021/bi400561e), we describe the use of the two-plasmid system to produce and characterize, in membranes and in purified states, an active heterodimeric cytochrome <i>bc</i>₁ variant with unusual intermonomer electron transfer properties

New Method for the Spin Quantitation of [4Fe?4S] + Clusters with S = 3 / 2 . Application to the FS0 Center of the NarGHI Nitrate Reductase from Escherichia coli

International audienceIn conventional analyses of g ≈ 5 signals given by [4Fe−4S]+ clusters with S = 3/2, the effective g values that cannot be measured in the electron paramagnetic resonance (EPR) spectrum are deduced from rhombograms calculated by assuming that the g̃ matrix is isotropic with gx = gy = gz = 2.00. We have shown that when the two low-field peaks corresponding to the Kramers doublets are visible in the spectrum, a new, independent piece of information about the system can be obtained by studying the temperature dependence of the ratio of the area under these peaks. By applying this method to the g ≈ 5 signals displayed by NarGHI nitrate reductase, we were able to determine all the parameters of the spin Hamiltonian of FS0 centers with S = 3/2 and to measure accurately their number. Our results indicate that simple analyses based on the assumption of an isotropic g̃ matrix can give rise to very large errors

The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qo site of bacterial cytochrome bc1

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1

Biogenesis of a Respiratory Complex Is Orchestrated by a Single Accessory Protein

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