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 CO 2 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 O 2 . 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.Chlamydomonas reinhardtii is a unicellular, soil-dwelling, photosynthetic green alga that has a diversity of fermentation pathways, inferred from the full genome sequence (1, 2). It uses these pathways for ATP production during anoxia, catabolizing starch and other intracellular carbon substrates into the predominant fermentation products formate, acetate, ethanol, CO 2 , and molecular hydrogen (H 2 ) in what is classified as heterofermentation (2-7). These metabolic pathways would be active primarily at night when high rates of respiration and the absence of photosynthetic O 2 evolution cause the rapid establishment of anoxia (8), especially in soil environments with high concentrations of microbes. Moreover, C. reinhardtii can balance the use of the tricarboxylic acid cycle with fermentation when the rate of respiratory O 2 consumption exceeds the rate of photosynthetic O 2 evolution (3, 4, 9). The catabolism of intracellular carbon stores during anoxic acclimation is a key component of C. reinhardtii metabolism as this alga does not appear to effectively assimilate extracellular sugars. Acquiring a better understanding of cellular metabolisms in algae such as C. reinhardtii under various conditions will facilitate the development o...

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…This figure was modified from Grossman et al (2011). fermentation metabolism (Dubini et al, 2009;Grossman et al, 2011;Philipps et al, 2011;Catalanotti et al, 2012). For example, in the Chlamydomonas hydEF-1 mutant (Dubini et al, 2009), pyruvate metabolism is redirected to the reverse tricarboxylic acid reactions, while in the pfl1 mutant (Philipps et al, 2011;Catalanotti et al, 2012), there is a marked increase in lactate production and a smaller increase in ethanol synthesis; both of these metabolites are linked to NADH reoxidation (Fig.…”

“…In wild-type cells (black arrows), the major fermentative products are formate, acetate, and ethanol, with CO 2 and H 2 emitted as minor products. The metabolic pathway that leads to the fermentative production of succinate is unveiled in the hydEF-1 mutant (Dubini et al, 2009) and is depicted in the figure in green. An increase in the production of lactate, which is almost undetectable in fermenting wild-type cells, has been observed in the pfl1 mutants (Philipps et al, 2011;Catalanotti et al, 2012) and is highlighted in orange.…”

“…Some enzymes that function in these pathways include pyruvate:ferredoxin oxidoreductase (PFR), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate formate lyase (PFL), alcohol dehydrogenase (ADH), phosphate acetyltransferase (PAT), acetate kinase (ACK), and the two [FeFe] hydrogenases (HYDA1 and HYDA2) and their maturation proteins, HYDG and HYDEF (Posewitz et al, 2004;Atteia et al, 2006;Ghirardi et al, 2007;Mus et al, 2007;Hemschemeier et al, 2008;Grossman et al, 2011). The anaerobic activities of these and other enzymes result in the secretion of organic acids (formate, lactate, malate, acetate, and succinate) and alcohols (ethanol and glycerol) as well as the evolution of H 2 and CO 2 (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987;Tsygankov et al, 2002;Kosourov et al, 2003;Mus et al, 2007;Dubini et al, 2009).…”

Unraveling Vitamin B12-Responsive Gene Regulation in Algae

“…Transcript abundance is shown when C. reinhardtii is grown in the presence or absence of vitamin B 12 (1,000 ng L 21 ). Transcripts were quantified using RT-qPCR with RACK1 as the reference gene (Mus et al, 2007;Dubini et al, 2009). For each gene, the result was normalized by expression in the absence of B 12 .…”

“…prior to B 12 addition). Transcript expression was quantified and normalized using RACK1 as a housekeeping gene (Mus et al, 2007;Dubini et al, 2009). Values are means and error bars denote the range (n = 2).…”