A mutant in the ADH1 gene of Chlamydomonas reinhardtii elicits metabolic restructuring during anaerobiosis

The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants

Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes

Access to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2 to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.Biotechnology and Biological Sciences Research Council (Great Britain) (Grants BB/L011484/1 and BB/L011476/1)National Science Foundation (U.S.) (Grant 1331098

Inhibitor studies on non-photochemical plastoquinone reduction and H2 photoproduction in Chlamydomonas reinhardtii

International audienc

Characterization of Nda2, a Plastoquinone-reducing Type II NAD(P)H Dehydrogenase in Chlamydomonas Chloroplasts

International audienc

Exploring the Alternatives of Biological Nitrogen Fixation

Most biological nitrogen fixation (BNF) results from the activity of the molybdenum nitrogenase (Mo-nitrogenase, Nif), an oxygen-sensitive metalloenzyme complex found in all known diazotrophs. Two alternative forms of nitrogenase, the vanadium nitrogenase (V-nitrogenase, Vnf) and the iron-only nitrogenase (Fe-only nitrogenase, Anf) have also been identified in the genome of some organisms that encode for Nif. It has been suggested that alternative nitrogenases were responsible for N2-fixation on early Earth because oceans were depleted of bioavailable Mo. Results of recent phylogenetic- and structure-based studies suggest, however, that such an evolutionary path is unlikely, and favor a new model for a stepwise evolution of nitrogenase where the V-nitrogenase and the Fe-only nitrogenase are not the ancestor of the Mo-nitrogenase. Rather, Mo-nitrogenase emerged within the methanogenic archaea and then gave rise to the alternative forms suggesting they arose later in response to the availability of fixed N2 and local environmental factors that influenced metal availability. This review summarizes the current state of knowledge on (1) the biochemistry of these complex systems highlighting the common and specific structural features and catalytic activities of the enzymes, (2) the recent progress in defining the discrete set of genes associated to N2-fixation and the regulatory features that coordinate the differential expression of genes in response to metal availability, and (3) the diverse taxonomic and phylogenic distribution of nitrogenase enzymes and the evolutionary history of BNF from the perspective of metal content and metal availability

Activité NAD(P)H plastoquinone oxydoréductase et photo-production d'hydrogène au sein des thylacoïdes de l'algue verte Chlamydomonas reinhardtii

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF