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Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês

10.1074/jbc.m803917200

ISSN

1083-351X

Autores

Alexandra Dubini, Florence Mus, Michael Seibert, Arthur Grossman, Matthew C. Posewitz,

Tópico(s)

Hybrid Renewable Energy Systems

Resumo

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. 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. Chlamydomonas reinhardtii is a unicellular, soil-dwelling, photosynthetic green alga that has a diversity of fermentation pathways, inferred from the full genome sequence (1Merchant S.S. Prochnik S.E. Vallon O. Harris E.H. Karpowicz S.J. Witman G.B. Terry A. Salamov A. Fritz-Laylin L.K. Marechal-Drouard L. Marshall W.F. Qu L.H. Nelson D.R. Sanderfoot A.A. Spalding M.H. Kapitonov V.V. Ren Q. Ferris P. Lindquist E. Shapiro H. Lucas S.M. Grimwood J. Schmutz J. Cardol P. Cerutti H. Chanfreau G. Chen C.L. Cognat V. Croft M.T. Dent R. Dutcher S. Fernandez E. Fukuzawa H. Gonzalez-Ballester D. Gonzalez-Halphen D. Hallmann A. Hanikenne M. Hippler M. Inwood W. Jabbari K. Kalanon M. Kuras R. Lefebvre P.A. Lemaire S.D. Lobanov A.V. Lohr M. Manuell A. Meier I. Mets L. Mittag M. Mittelmeier T. Moroney J.V. Moseley J. Napoli C. Nedelcu A.M. Niyogi K. Novoselov S.V. Paulsen I.T. Pazour G. Purton S. Ral J.P. Riano-Pachon D.M. Riekhof W. Rymarquis L. Schroda M. Stern D. Umen J. Willows R. Wilson N. 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It uses these pathways for ATP production during anoxia, catabolizing starch and other intracellular carbon substrates into the predominant fermentation products formate, acetate, ethanol, CO2, and molecular hydrogen (H2) in what is classified as heterofermentation (2Grossman A.R. Croft M. Gladyshev V.N. Merchant S.S. Posewitz M.C. Prochnik S. Spalding M.H. Curr. Opin. Plant Biol. 2007; 10: 190-198Crossref PubMed Scopus (131) Google Scholar, 3Kosourov S. Seibert M. Ghirardi M.L. Plant Cell Physiol. 2003; 44: 146-155Crossref PubMed Scopus (219) Google Scholar, 4Ghirardi M.L. Posewitz M.C. Maness P.-C. Dubini A. Yu J. Seibert M. Annu. Rev. Plant Biol. 2007; 58: 71-91Crossref PubMed Scopus (303) Google Scholar, 5Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 7Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar). These metabolic pathways would be active primarily at night when high rates of respiration and the absence of photosynthetic O2 evolution cause the rapid establishment of anoxia (8Steunou A.S. Bhaya D. Bateson M.M. Melendrez M.C. Ward D.M. Brecht E. Peters J.W. Kuhl M. Grossman A.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2398-2403Crossref PubMed Scopus (165) Google Scholar), 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 O2 consumption exceeds the rate of photosynthetic O2 evolution (3Kosourov S. Seibert M. Ghirardi M.L. Plant Cell Physiol. 2003; 44: 146-155Crossref PubMed Scopus (219) Google Scholar, 4Ghirardi M.L. Posewitz M.C. Maness P.-C. Dubini A. Yu J. Seibert M. Annu. Rev. Plant Biol. 2007; 58: 71-91Crossref PubMed Scopus (303) Google Scholar, 9Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (887) Google Scholar). 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 of physiological models that predict metabolic circuits and the interactions among these circuits. Additionally, the secretion of fermentative metabolites by C. reinhardtii (and other algae) likely has significant impacts on the population dynamics of microbial consortia in environments inhabited by C. reinhardtii. There is also the potential to leverage the unique metabolic flexibility of C. reinhardtii for the production of valuable metabolites such as H2, organic acids, and ethanol as renewable bioenergy carriers (4Ghirardi M.L. Posewitz M.C. Maness P.-C. Dubini A. Yu J. Seibert M. Annu. Rev. Plant Biol. 2007; 58: 71-91Crossref PubMed Scopus (303) Google Scholar, 10Kruse O. Rupprecht J. Mussgnug J.H. Dismukes G.C. Hankamer B. Photochem. Photobiol. Sci. 2005; 4: 957-970Crossref PubMed Scopus (248) Google Scholar, 11Hankamer B. Lehr F. Rupprecht J. Mussgnug J.H. Posten C. Kruse O. Physiol. Plant. 2007; 131: 10-21Crossref PubMed Scopus (173) Google Scholar). Although studies of C. reinhardtii metabolism have already significantly advanced our understanding of cellular responses to anoxia (5Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 7Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 12Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar), additional research efforts are required to gain further insights into the proteins involved in adaptation and acclimation of the cells to anaerobiosis, the modulation of cellular metabolite levels under these conditions, and the accurate localization of fermentation pathways and proteins to specific subcellular compartments. The availability of the C. reinhardtii genome sequence, combined with high throughput "omics"-based approaches, will be critical in this effort. Moreover, the use of specific mutant strains can help establish the foundation for a more comprehensive understanding of how cells adjust metabolite fluxes when specific reactions are blocked. A number of studies have demonstrated that C. reinhardtii can rapidly acclimate to anaerobiosis by shifting to fermentative metabolisms (5Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 7Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, 12Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar, 13Kreutzberg K. Physiol. Plant. 1984; 61: 87-94Crossref Scopus (74) Google Scholar, 14Ohta S. Miyamoto K. Miura Y. Plant Physiol. 1987; 83: 1022-1026Crossref PubMed Google Scholar). The catabolism of pyruvate to acetyl-CoA in C. reinhardtii may proceed via fermentation pathways that use either PFL1 (pyruvate formate-lyase) (5Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar, 15Hemschemeier A. Jacobs J. Happe T. Eukaryot. Cell. 2008; 7: 518-526Crossref PubMed Scopus (47) Google Scholar) or PFR1 (pyruvate ferredoxin oxidoreductase) (5Atteia A. van Lis R. Gelius-Dietrich G. Adrait A. Garin J. Joyard J. Rolland N. Martin W. J. Biol. Chem. 2006; 281: 9909-9918Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 12Hemschemeier A. Happe T. Biochem. Soc. Trans. 2005; 33: 39-41Crossref PubMed Scopus (61) Google Scholar). The acetyl-CoA is further metabolized to ethanol and/or acetate, and the reduced ferredoxin generated by PFR1 activity is putatively oxidized by a number of redox proteins, including one or both of the two C. reinhardtii [FeFe] hydrogenases (HYDA1 and HYDA2) (16Forestier M. King P. Zhang L. Posewitz M. Schwarzer S. Happe T. Ghirardi M.L. Seibert M. Eur. J. Biochem. 2003; 270: 2750-2758Crossref PubMed Scopus (210) Google Scholar, 17Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (212) Google Scholar), which are localized to the chloroplast stroma (18Happe T. Mosler B. Naber J.D. Eur. J. Biochem. 1994; 222: 769-774Crossref PubMed Scopus (148) Google Scholar, 19Roessler P.G. Lien S. Plant Physiol. 1984; 75: 705-709Crossref PubMed Google Scholar). A recently isolated C. reinhardtii mutant, hydEF-1, is devoid of hydrogenase activity (20Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 21Posewitz M.C. King P.W. Smolinski S.L. Smith R.D. Ginley A.R. Ghirardi M.L. Seibert M. Biochem. Soc. Trans. 2005; 33: 102-104Crossref PubMed Scopus (78) Google Scholar) because of disruption of an [FeFe] hydrogenase maturase, which is required for proper enzyme assembly (20Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 21Posewitz M.C. King P.W. Smolinski S.L. Smith R.D. Ginley A.R. Ghirardi M.L. Seibert M. Biochem. Soc. Trans. 2005; 33: 102-104Crossref PubMed Scopus (78) Google Scholar, 22Posewitz M.C. Mulder D.W. Peters J.W. Curr. Chem. Biol. 2008; 2: 178-199Google Scholar, 23McGlynn S.E. Ruebush S.S. Naumov A. Nagy L.E. Dubini A. King P.W. Broderick J.B. Posewitz M.C. Peters J.W. J. Biol. Inorg. Chem. 2007; 12: 443-447Crossref PubMed Scopus (101) Google Scholar, 24Bock A. King P.W. Blokesch M. Posewitz M.C. Adv. Microb. Physiol. 2006; 51: 1-71Crossref PubMed Scopus (299) Google Scholar); consequently, neither of the hydrogenases are active in the mutant strain. In this study we use molecular and physiological approaches to examine dark, anoxic acclimation of the C. reinhardtii hydEF-1 mutant. Interestingly, the mutant secretes much higher levels of succinate during anoxia than the parental strain. Furthermore, transcripts encoding PYC1 (pyruvate carboxylase) and MME4 (a malic enzyme) increase significantly in the mutant relative to the parental strain during anaerobiosis. Both of these proteins have the ability to carboxylate pyruvate, providing precursors for the fermentative production of succinate. Additionally, transcripts encoding fumarase (FUM1 and FUM2) and FMR1 (fumarate reductase), enzymes that catalyze the final steps of succinate synthesis, are elevated in the hydEF-1 strain. Utilization of these pathways for succinate synthesis would result in NADH reoxidation, which would sustain additional cycles of glycolysis and explain both the relative increase in succinate and decrease in CO2 production during anaerobiosis in the mutant relative to the parental strain. Microarray studies were also performed and reveal that several transcripts encoding proteins associated with cellular redox functions and other aspects of anaerobic metabolism are differentially regulated in the mutant relative to the parental strain. These findings are discussed with respect to the identification of potential new fermentation pathways and the flexibility of whole-cell metabolism in C. reinhardtii under dynamic environmental conditions. Strains and Growth Conditions-C. reinhardtii CC-425 (cw15, sr-u-60, arg7-8, mt+) and hydEF-1 mutant (derived from CC-425) cells were grown in Tris/acetate/phosphate medium (TAP) 2The abbreviations used are: TAP, Tris/acetate/phosphate medium; qPCR, quantified by reverse transcriptase real time PCR; HPLC, high pressure liquid chromatography; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PCK, phosphoenolpyruvate carboxylase; MDH, malate dehydrogenase; FUM, fumarase; FMR, fumarate reductase; MME, malic enzyme. (pH 7.2), supplemented with 200 mg·liter-1 arginine for CC-425. Algal cultures were maintained at 25 °C, vigorously bubbled with air enriched with 3% CO2, stirred using a magnetic stir bar, and illuminated with continuous light of 80 μmol photon m-2 s-1 photosynthetically active radiation at the surface of 1-liter Roux culture bottles (255 × 55 × 120 mm), in which cell densities ranged from 1 × 105 to 3 × 106 cells/ml of culture. Chlorophyll Measurements-Chlorophyll a and b content were determined spectrophotometrically in 95% ethanol (25Harris E.H. The Chlamydomonas Sourcebook. A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego1989: 780-785Google Scholar). Anaerobic Induction of Liquid Cell Suspensions-C. reinhardtii cultures were grown on TAP medium to ∼16-24 μg·ml-1 total chlorophyll, centrifuged (500 ml of cells) at 3,000 × g for 1 min, and the cell pellet resuspended in one-tenth volume (50 ml) of anaerobic induction buffer containing 50 mm potassium phosphate (pH 7.2) and 3 mm MgCl2 (26Ghirardi M. Togasaki R. Seibert M. Appl. Biochem. Biotech. 1997; 63-5: 141-151Crossref Google Scholar). The cells were then transferred to a sealed anaerobic vial in the dark and flushed with argon for 30 min. For measuring H2 and O2 production rates, 200 μl of cell cultures were placed in a Clark-type oxygen electrode assay chamber as described previously (6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 20Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Clark-type oxygen electrodes were used simultaneously to measure light-induced H2 and O2 production rates. To measure fermentative H2 production, 400 μl of head space gas was withdrawn from the sealed anaerobic vials and analyzed by gas chromatography (Hewlett Packard 5890 series II) using a Supelco column (60/80 mol sieve 5A 6 feet × 1/8 inch) coupled to a TCD detector. H2 remaining in solution was quantified using the Clark-type oxygen electrode described above without illumination of the sample chamber. CO2 Measurement-CO2 levels were below detection limits in the serum vial head space of anaerobically acclimated cells. Therefore, following anaerobic induction, 1 ml of anoxic cells was transferred in a gas-tight syringe to a sealed vial into which 1 ml of 1 m HCl was added. The acidified cell suspension was vigorously shaken to liberate CO2, which was quantified by gas chromatography (Hewlett Packard 5890 series II) using a Supelco column (80/100 PORAPAK N 6 feet × 1/8 inch × 2.1 mm) coupled to a TCD detector. Metabolite Analysis-Organic acid analysis was performed by liquid chromatography (Hewlett Packard Series 1050 HPLC) using an Aminex HPX-87H (300 × 7.8 mm) ion exchange column. Dark-adapted cells were collected at the indicated time points and centrifuged (10,000 × g for 1 min), and the supernatant was transferred to a new vial and frozen in liquid N2 for subsequent analysis. Samples were thawed, centrifuged, and filtered prior to HPLC analysis. One hundred μl of cell culture supernatant was injected onto the column and eluted using 8 mm filtered sulfuric acid (J. T. Baker, Inc.) as the mobile phase at a flow rate of 0.5 ml·min-1 at 45 °C. Retention peaks were recorded using Agilent ChemStation software, and quantifications were performed by comparisons with known amounts of standard for each of the organic acids. Ethanol was measured using a YSI 2700 SELECT electrochemical probe (YSI Inc., Yellow Springs, OH). Identical supernatants were used for metabolite and ethanol analysis; 10 μl of the supernatant was required for ethanol analysis. Extraction of RNA-Total RNA was isolated using the Plant RNA reagent, as described by the manufacturer (Invitrogen). Approximately 40 μg of isolated RNA was treated with 5 units of RNase-free DNase (Ambion, Austin, TX) for 30 min at room temperature. The Qiagen RNeasy MinElute kit (Qiagen, Valencia, CA) was used to purify total RNA. The A260 of the eluted RNA was measured, and 4 μg of purified RNA was reserved to prepare labeled samples for microarray analysis. Reverse Transcriptase Real Time PCR (qPCR)-The abundance of specific transcripts in total mRNA from each sample was quantified by reverse transcriptase real time PCR, designated qPCR, using the Engine Opticon system (Bio-Rad). First strand cDNA synthesis was primed on purified total RNA using specific primers for each C. reinhardtii gene of interest. The specific primers (at 0.5 μm each; supplied by IDT) were annealed to 250 ng of DNA-free total RNA and extended for 1 h at 55 °C using 200 units of the reverse transcriptase Superscript III (Invitrogen). Four μl of the single-stranded cDNA from the reverse transcriptase reaction (final volume, 20 μl) was used as the template for real time PCR amplifications, which were performed using the DyNAmo HS SYBR green reverse transcription-PCR kit according to the manufacturer's instructions (Finnzymes, Woburn, MA). Specific primers (0.3 μm) used for amplification were designed to generate amplicons of 100-200 nucleotides. Amplifications were performed using a Bio-Rad iCycler iQ detection system and the following cycling parameters: an initial single step at 95 °C for 10 min (denaturation) followed by 40 cycles of (a) 94 °C for 30 s (denaturation), (b) 56 °C for 45 s (annealing), and (c) 72 °C for 30 s (elongation). Following the 40 cycles, a final elongation/termination step was performed at 72 °C for 10 min. Primers used for qPCR are shown in supplemental Table 1, and were designed using Primer3 software (available on line). The relative expression ratio of a target gene was calculated based on the 2-ΔΔCT method (27Livak K.J. Schmittgen T.D. Methods (San Diego). 2001; 25: 402-408Crossref PubMed Scopus (124148) Google Scholar), using the average cycle threshold (CT) calculated from triplicate measurements. Relative expression ratios from three independent experiments (different experimental replicates) are reported. The level of accumulation of the RACK1 transcript was used as a normalization control because it shows near constant expression under the conditions used in these experiments. Microarray Fabrication-Microarrays were fabricated at the Stanford Functional Genomics Facility at Stanford University, as described previously (6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Labeling and Purification of Reverse-transcribed cDNAs-Labeling and purification of reverse-transcribed cDNAs were performed as described previously (28Eberhard S. Jain M. Im C.S. Pollock S. Shrager J. Lin Y. Peek A.S. Grossman A.R. Curr. Genet. 2006; 49: 106-124Crossref PubMed Scopus (34) Google Scholar). Briefly, 4 μg of purified RNA was adjusted to 4 μl with sterile MilliQ-treated water. One μl of oligo(dT)-(V) (2 μg·μl-1) was heated for 10 min at 70 °C and quickly chilled on ice. The following reagents were added sequentially to the reaction mixture: 2 μl of 5× superscript buffer; 1 μl of 0.1 m dithiothreitol; 0.2 μl of 50× dNTPs (5 mm dATP, dCTP, and dGTP and 10 mm dTTP); 1 μl of Cy3- or Cy5-dUTP; and 0.8 μl of Superscript III (200 units·μl-1). The final reaction volume was 10 μl. After allowing the reaction to proceed at 42 °C for 2 h, an additional aliquot of 0.5 μl of Superscript III was added, and the reaction was continued for an additional 1 h at 50 °C. The reaction was stopped by the addition of 0.5 μl of 500 mm EDTA and 0.5 μl of 500 mm NaOH, and the solution was incubated at 70 °C for 10 min. Neutralization of the reaction mixture was achieved by adding 0.5 μl of 500 mm HCl. The QIAquick PCR purification kit (Qiagen, Valencia, CA) was used to purify the labeled cDNA as described previously (6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Hybridization to the Oligonucleotide Array-Pre-hybridization and hybridization of fluorescent-labeled probes and washing of the arrays were as described previously (6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 28Eberhard S. Jain M. Im C.S. Pollock S. Shrager J. Lin Y. Peek A.S. Grossman A.R. Curr. Genet. 2006; 49: 106-124Crossref PubMed Scopus (34) Google Scholar). Detailed and updated versions of the protocols used for RNA labeling, slide pre-hybridization, hybridization, and washing can be downloaded from the Chlamydomonas Center website. Scanning, Quantification, and Analysis of the Slides-Microarray slides were scanned for Cy5 and Cy3 fluorescent signals using a GenePix 4000B scanner (Molecular Devices) as described previously (6Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Spot positions were defined using SpotReader (Niles Scientific). Analyses of the data were performed using GeneSpring 6.1 (Agilent Technologies, Foster City, CA). Images of the fluorescence at 532 nm for Cy3 and 635 nm for Cy5 were recorded and analyzed from the complete array sets (three experimental replicates for the time point 0.5 h, four experimental replicates for the time points 2 and 4 h, and three slides per time point for each experimental replicate, with two copies of each cDNA per slide). Error models were computed based on replicates. Signal ratios were considered to meet threshold criteria if they passed Student's t test for significance with a p value of ≤0.05. Metabolite Analyses-The C. reinhardtii hydEF-1 mutant lacks hydrogenase activity because of disruption of the HYDEF gene, which encodes a hydrogenase maturase (20Posewitz M.C. King P.W. Smolinski S.L. Zhang L. Seibert M. Ghirardi M.L. J. Biol. Chem. 2004; 279: 25711-25720Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). This mutant was used to investigate the physiological responses of C. reinhardtii in the absence of H2 metabolism during anaerobic acclimation. The fermentative products that accumulated in the medium during anoxia in both cultures mutant and parental control are shown in Table 1. Four significant differences observed between mutant and parental cells were as follows: (a) the absence of H2 production in the mutant relative to the parental strain; (b) a marked difference in accumulation of CO2 between the two strains; (c) a slight reduction in the rate of accumulation of formate, acetate, and ethanol in the mutant relative to the parental strain; and (d) elevated production of succinate, only in the mutant. Rescue of the hydEF-1 mutant phenotype and restoration of the fermentation profile (along with H2 and CO2 production) observed in the parental strain was achieved by introduction of a functional copy of the HYDEF gene (data not shown). The complemented strain was not used as a reference in the microarray experiments as integration of the introduced HYDEF gene into the genome could disrupt another cellular function.TABLE 1Concentrations of fermentation products secreted by the Chlamydomonas hydEF-1 mutant and the parental strain, CC-425, following exposure to dark anaerobiosis at the indicated timesFermentation productsTimeCO2H2Formic acidSuccinic acidAcetic acidEthanolhmmol/literCC-4250.5−0.05 ± 0.090.02 ± 0.010.5 ± 0.2BDaBD means below detection.0.9 ± 0.20.2 ± 0.120.14 ± 0.020.03 ± 0.011.3 ± 0.10.1 ± 0.11.5 ± 0.10.7 ± 0.140.64 ± 0.080.06 ± 0.012.2 ± 0.10.1 ± 0.11.8 ± 0.31.1 ± 0.224NDbND means not determined.ND4.8 ± 0.20.1 ± 0.14.3 ± 0.31.9 ± 0.1hydEF-10.5−0.09 ± 0.01BD0.3 ± 0.20.1 ± 0.030.4 ± 0.2BD2−0.09 ± 0.02BD1.0 ± 0.10.2 ± 0.10.7 ± 0.20.4 ± 0.14−0.03 ± 0.01BD1.5 ± 0.10.4 ± 0.21.0 ± 0.20.7 ± 0.124NDND3.5 ± 0.21.2 ± 0.12.0 ± 0.20.8 ± 0.2a BD means below detection.b ND means not determined. Open table in a new tab Succinate is not detected in significant amounts in the medium of the parental culture but does accumulate in the mutant culture, particularly after longer periods (4 and 24 h) of anaerobiosis. CO2 levels do not increase significantly during acclimation to anaerobiosis in the mutant, whereas in parental cells CO2 levels steadily rise over the acclimation period. To account for succinate production and the near constant CO2 level in the mutant, we identified genes on the draft genome encoding enzymes associated with fermentative succinate production. These encoded enzymes were placed into metabolic pathways (Fig. 1) leading to the fermentative production of succinate, which requires the carboxylation of a three-carbon substrate and is consistent with diminished CO2 accumulation in the mutant relative to the parental strain. The proteins associated with the succinate pathways depicted in Fig. 1 are given in Table 2. Because hydrogenase activity is absent in the mutant, pyruvate flux via the pyruvate ferredoxin oxidoreductase (PFR1) pathway is anticipated to be altered because hydrogenase can no longer participate in PFR1/ferredoxin reoxidation. However, it should be noted that additional me

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