Florence Mus

Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways.

Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production.

FLEXIBILITY IN ANAEROBIC METABOLISM AS REVEALED IN A MUTANT OF CHLAMYDOMONAS REINHARDTII LACKING HYDROGENASE ACTIVITY

The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

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

Electron transfer pathways associated to oxygenic photosynthesis, including cyclic electron flow around photosystem I and chlororespiration, rely on non-photochemical reduction of plastoquinones (PQs). In higher plant chloroplasts, a bacterial-like NDH complex homologous to complex I is involved in PQ reduction, but such a complex is absent from Chlamydomonas plastids where a type II NAD(P)H dehydrogenase activity has been proposed to operate. With the aim to elucidate the nature of the enzyme-supporting non-photochemical reduction of PQs, one of the type II NAD(P)H dehydrogenases identified in the Chlamydomonas reinhardtii genome (Nda2) was produced as a recombinant protein in Escherichia coli and further characterized. As many type II NAD(P)H dehydrogenases, Nda2 uses NADH as a preferential substrate, but in contrast to the eukaryotic enzymes described so far, contains non-covalently bound FMN as a cofactor. When expressed at a low level, Nda2 complements growth of an E. coli lacking both NDH-1 and NDH-2, but is toxic at high expression levels. Using an antibody raised against the recombinant protein and based on its mass spectrometric identification, we show that Nda2 is localized in thylakoid membranes. Chlorophyll fluorescence measurements performed on thylakoid membranes show that Nda2 is able to interact with thylakoid membranes of C. reinhardtii by reducing PQs from exogenous NADH or NADPH. We discuss the possible involvement of Nda2 in cyclic electron flow around PSI, chlororespiration, and hydrogen production.

Limiting steps of hydrogen production in Chlamydomonas reinhardtii and Synechocystis PCC 6803 as analysed by light-induced gas exchange transients

In the cyanobacterium Synechocystis PCC 6803 and in the microalga Chlamydomonas reinhardtii, transient hydrogen photo-production is observed when cells are exposed to light in anoxia. We measured changes in H 2 , O 2 , and CO 2 concentrations using time-resolved mass spectrometry in wild-type and mutant strains of Chlamydomonas and Synechocystis. In both organisms, non-photochemical reduction of the plastoquinone pool is shown to contribute to the initial H 2 photo-production. This pathway, which does not produce O 2 , exhibits a low rate in normal conditions. From the effect of the uncoupler FCCP, we conclude that PS II-independent H 2 production in Chlamydomonas is limited by the trans-thylakoidal proton gradient. In Synechocystis, from the study of a mutant deficient in the NDH-1 complex (M55), we conclude that PS II-independent H 2 production is limited by recycling of NAD(P)H through the NDH-1 complex. Based on these conclusions, we propose strategies for optimising H 2 photo-production in these organisms.

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.

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.

Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes

ABSTRACT 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.

[FeFe]-hydrogenase maturation.

Hydrogenases are metalloenzymes that catalyze the reversible reduction of protons at unusual metal centers. This Current Topic discusses recent advances in elucidating the steps involved in the biosynthesis of the complex metal cluster at the [FeFe]-hydrogenase (HydA) active site, known as the H-cluster. The H-cluster is composed of a 2Fe subcluster that is anchored within the active site by a bridging cysteine thiolate to a [4Fe-4S] cubane. The 2Fe subcluster contains carbon monoxide, cyanide, and bridging dithiolate ligands. H-cluster biosynthesis is now understood to occur stepwise; standard iron-sulfur cluster assembly machinery builds the [4Fe-4S] cubane of the H-cluster, while three specific maturase enzymes known as HydE, HydF, and HydG assemble the 2Fe subcluster. HydE and HydG are both radical S-adenosylmethionine enzymes that interact with an iron-sulfur cluster binding GTPase scaffold, HydF, during the construction of the 2Fe subcluster moiety. In an unprecedented biochemical reaction, HydG cleaves tyrosine and decomposes the resulting dehydroglycine into carbon monoxide and cyanide ligands. The role of HydE in the biosynthetic pathway remains undefined, although it is hypothesized to be critical for the synthesis of the bridging dithiolate. HydF is the site where the complete 2Fe subcluster is formed and ultimately delivered to the immature hydrogenase protein in the final step of [FeFe]-hydrogenase maturation. This work addresses the roles of and interactions among HydE, HydF, HydG, and HydA in the formation of the mature [FeFe]-hydrogenase.

Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase

This article describes novel ways that algae may adjust metabolite trafficking when specific branches of fermentation metabolism are blocked. This rerouting of metabolites allows for continued glycolytic energy production under anoxic conditions, which is critical for the cell’s survival. Mechanisms associated with this reengineering of metabolism are almost completely unexplored. Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H2 production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

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 CO2 was significantly reduced, but fermentative H2 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.