References of "Emonds-Alt, Barbara"
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See detailA forward genetic screen to identify hydrogenase-deficient mutants in the unicellular green alga Chlamydomonas reinhardtii
Emonds-Alt, Barbara ULg; Godaux, Damien ULg; Cardol, Pierre ULg et al

Poster (2014, June 15)

The ability of the unicellular green alga Chlamydomonas reinhardtii to evolve molecular hydrogen (H2) is due to the presence of oxygen-sensitive Fe-hydrogenases (HydA1/2), expressed in anoxic conditions ... [more ▼]

The ability of the unicellular green alga Chlamydomonas reinhardtii to evolve molecular hydrogen (H2) is due to the presence of oxygen-sensitive Fe-hydrogenases (HydA1/2), expressed in anoxic conditions that drive the photosynthetic electron flow to reduce protons into H2. In order to identify new players involved in H2 photoproduction in Chlamydomonas, an insertion mutant library was generated using cassettes conferring resistance to hygromycin or paromomycin. Hydrogenase activity is physiologically relevant during a transition from dark anoxia to light. In dark anoxic conditions, the cellular redox poise is high and the photosynthetic electron transport chain is fully reduced. Upon illumination, hydrogenase activity allows the reoxidation of photosynthetic intersystem electron carriers until oxic conditions and carbon fixation ability are restored. We thus designed an in vivo fluorescence imaging screen based on the different kinetics of photosynthesis induction between wild type and hydrogenase-deficient mutants [1]. At this stage, three putative hydrogenase mutants have been identified on 10,000 transformants. Molecular characterization of the insertion site of the resistance cassette by TAIL-PCR and genetic analyses of the linkage between the antibiotic resistance and the fluorescence phenotype showed that one mutant was untagged with the resistance while two tagged mutants were deficient for the HydG assembly factor. [less ▲]

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See detailLack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.
Plancke, Charlotte; Vigeolas, Hélène ULg; Hohner, Ricarda et al

in The Plant journal : for cell and molecular biology (2014), 77(3), 404-417

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the ... [more ▼]

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by 14 N/15 N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in beta-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat. [less ▲]

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See detailA novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Berne, Nicolas ULg et al

in International Journal of Hydrogen Energy (2013), 38

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes <br />Received 30 August 2012 belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases ... [more ▼]

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of enzymes <br />Received 30 August 2012 belonging to various fermentative pathways. Among them, oxygen-sensitive hydrogenases <br />Received in revised form (HydA1/2) catalyze the synthesis of molecular hydrogen from protons and reduced ferre- <br />12 November 2012 doxin in the stroma. In this work, by analyzing wild type and mutants affected in H2 <br />Accepted 16 November 2012 production, we show that maximal PSII photosynthetic electron transfer during the first <br />Available online 21 December 2012 seconds of illumination after a prolonged dark-anaerobiosis period is linearly related to <br />hydrogenase capacity. Based on the specific chlorophyll fluorescence induction kinetics <br />Keywords: typical of hydrogenase-deficient mutants, we set up an in vivo fluorescence imaging <br />Chlamydomonas reinhardtii screening protocol allowing to isolate mutants impaired in hydrogenase expression or <br />Anaerobic photosynthesis activity, as well as mutants altered in related metabolic pathways required for energy <br />Hydrogenase production in anaerobiosis. Compared to previously described screens for mutants <br />Chlorophyll fluorescence impaired in H2 production, our screening method is remarkably fast, sensitive and non- <br />Microalgae invasive. Out of 3000 clones from a small-sized insertional mutant library, five mutants <br />Hydrogen photoproduction were isolated and the most affected one was analyzed and shown to be defective for the <br />hydrogenase HydG assembly factor. [less ▲]

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See detailLight induced photosynthetic electron transfer upon anaerobiosis in Chlamydomonas: Kinetics, electron sinks and setup of a fluorescence screen to identify new players
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Alric, Jean et al

Conference (2012, June 15)

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from ... [more ▼]

In Chlamydomonas reinhardtii, prolonged anaerobiosis leads to the expression of various fermentative pathways. Among them, oxygen-sensitive hydrogenases (hyd) catalyze the reduction of protons from reduced ferredoxin resulting in the production of molecular hydrogen. In this work, light-induced photosynthetic electron transfer after a prolonged dark-anaerobiosis period was studied by following the kinetics of chlorophyll fluorescence emission, P700 oxidation and proton-motive force formation and consumption during the first 3 seconds of illumination. We show that during the induction of photosynthesis, an hyd-dependent photosynthetic electron transfer operates at a maximal rate of 110 electrons per photosystem per second, that is about half the one measured in aerobiosis. The implication in this process of components of the linear, cyclic and chlororespiratory electron transfer pathways, as well as various electron sinks, are investigated thanks to the availability of mutants. In a next step, we screen an insertional mutant library (~3000 clones) on the basis of the fluorescence induction kinetics upon a shift from dark-anaerobiosis to light. Five mutants display the signature of mutants deficient for NADPH:PQ oxidoreductase or hyd activities. In particular, one is defective for hydrogenase HydG assembly factor. This mutant behaves exactly has the hydEF mutant, thus confirming that in vivo both the assembly factors are required for an efficient hydrogenase activity. [less ▲]

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See detailInsertional mutagenesis to select mutants for modified hydrogen photoproduction in Chlamydomonas reinhardtii
Godaux, Damien ULg; Emonds-Alt, Barbara ULg; Cardol, Pierre ULg et al

Poster (2011, May 17)

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases ... [more ▼]

The unicellular green alga Chlamydomonas reinhardtii has evolved the ability to redirect electrons from the photosynthetic chain to drive hydrogen production via chloroplast oxygen-sensitive hydrogenases. This process occurs under anaerobic conditions and provides a biological basis for solar-driven hydrogen production. Nevertheless, the yield is a major limitation for an economic viability and fundamental knowledge is still needed in order to have a better understanding of the process. In 2000, Melis and co-worker defined a protocol allowing a sustainable hydrogen production in sulfur deprivation condition. By adjustment of an existent protocol called the Winkler test, we are trying to isolate mutants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). This kind of mutants could be able to reach anoxia needed for hydrogenases activity without the stressful impact of sulfur deprivation. An insertional mutagenesis of Chlamydomonas has been carried out with an hygromycin resistance cassette and about 2500 transformants have generated and screened by the adapted Winkler test. We have isolated several oxygen-consuming mutants and the most promising one is subject to functional, molecular and genetic characterization. To discover new genes involved in hydrogenases activity, we are also planning to screen the same insertional library for mutants with attenuated levels of hydrogen photoproduction, using sensitive chemochromic sensor films which turn in blue in presence of hydrogen. We are currently making the chemochromic sensor WO3 films by dip-coating which is on the brink of being useable. [less ▲]

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