Flexibility in anaerobic metabolism as revealed in a mutant of Chlamydomonas reinhardtii lacking hydrogenase activity

Abstract

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 malate-forming 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.

FIGURE 1.

Anaerobic metabolic pathways in C. reinhardtii. Pathways at the upper right are proposed for succinate production and NADH reoxidation in the hydEF-1 mutant. Proteins encoded in the C. reinhardtii genome that are associated with the metabolisms depicted include the following: ACK, acetate kinase; ADH, bifunctional acetylaldehyde/alcohol dehydrogenase (alcohol dehydrogenase only in the case of the PDC pathway); FDX, ferredoxin; FMR, fumarate reductase; FUM, fumarase; HYD, hydrogenase; MDH, malate dehydrogenase; MME, malic enzyme; PAT, phosphate acetyltransferase; PDC, pyruvate decarboxylase; PEPC, phosphoenolpyruvate carboxylase; PFL, pyruvate formate-lyase; PFR, pyruvate-ferredoxin oxidoreductase; PYC, pyruvate carboxylase; PYK, pyruvate kinase. Known or putative cellular localizations of the proteins depicted are coded by color as follows: green for the chloroplast, red for the mitochondrion, orange for dual chloroplast/mitochondrion localization, blue for cytoplasm, and black for unknown. The cellular localizations of MDH1, -3, and -4, ACK2, PFL, and PAT1 were experimentally determined by Allmer et al. (46) and Atteia et al. (5), respectively. The cellular localizations of the other enzymes were determined using ChloroP, TargetP, and Predotar softwares and are highly speculative. When possible a final predicted localization was determined by doing an average of all predictions.

FIGURE 2.

Relative level of selected transcripts associated with pyruvate metabolism determined by qPCR at the indicated time of dark, anaerobic acclimation in hydEF-1 and the parental strain CC-425. Changes in indicated transcript levels following exposure of cells to dark, anaerobic conditions (0.5, 2, and 4 h) are presented as an n-fold change (arbitrary units, a.u.) relative (rel.) to RNA levels at time 0 (just prior to transfer and vigorously oxygenated). The results are normalized to RACK1 transcript levels, which remained constant over the course of the experiment. The results show the mean ± S.D. (error bars) for data from three biological (two technical repetitions for each biological replicate) qPCR replicates.

FIGURE 3.

Relative level of selected transcripts associated with succinate accumulation determined by qPCR at the indicated time of dark, anaerobic acclimation in hydEF-1 and the parental strain CC-425. Changes in transcript levels following exposure of the cells to dark, anaerobic conditions (0.5, 2, and 4 h) are presented as an n-fold change (arbitrary units, a.u.) relative (rel.) to RNA levels at time 0 (just prior to transfer and vigorously oxygenated). The results are normalized to RACK1 transcript levels, which remained constant over the course of the experiment. The results show the mean ± S.D. (error bars) for data from three biological (two technical repetitions for each biological replicate) qPCR replicates.