Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii Thomas Happe and Annette Kaminski Botanisches Institut der Universita ¨ t Bonn, Germany Chlamydomonas reinhardtii, a unicellular green alga, co n- tains a hydrogenase enzyme, which is induced by anaer- obic adaptation of the cells. Using the suppression subtractive hybridizatio n (SSH) approach, the d ifferential expression of genes under anaerobiosis was analyzed. A PCR f ragment with similarity t o the genes of ba cterial Fe-hydrogenases was isolated and used to screen an anaerobic c DNA expression librar y of C. reinhardtii.The cDNA sequence of hydA contains a 1494-bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa. The t ranscription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells. The deduced amino-acid sequence corresponds to two polypeptide sequences determined by sequence analysis of the isolated native p rotein. T he Fe-hydrogenase contains a short transit peptide o f 56 a mino acids, which routes the hydrogenase to the chloroplast stroma. The isolated protein belongs to a new class of Fe-hydrogenases. All four cysteine residues and 12 other a mino acids, which are s trictly c onserved i n t he active site (H-cluster) of Fe-hydrogenases, have been identified. The N-terminus of the C. reinhardtii protein is markedly truncated compared to other no nalgal Fe-hydrogenases. Further conserved cysteines that c oordinate additional F e–S-cluster i n other Fe-hydrogenases are missing. Ferredoxin PetF, the natural electron donor, links the hydrogenase from C. reinhardtii to the photosynthetic electron transport chain. The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme. Keywords: anaerobic adaptation; Chlamydomonas rein- hardtii; Fe-hydrogenase; hydrogen evolution; suppression subtractive hybridization. Green a lgae respond to anaerobic stress by s witching the oxidative pathway to a fermentative metabolism. The fermentation of organic compounds is associated with hydrogen evolution. The key enzyme hydrogenase, which is synthesized only after an anaerobic adaptation, catalyzes the reversible reduction of protons to molecular hydrogen. Hydrogenases are found in nearly all taxonomic groups of pr okaryotes [1,2] and some unicellular eukaryotic organisms [3,4]. With respect to the m etal composition in the active center, hydrogenases are divided into three classes: NiFe-hydrogenases [5,6], Fe-hydrogenases [7], and the hydrogenases without nickel and iron atoms, which were found only in archaea [8,9]. Fe-hydrogenases are characterized in h ydrogen-produc- ing anaerobic microorganisms and protozoa [3,10–13]. They are known for their CO sensitivity and an enzyme activity that is 100-fold higher than the activity of the NiFe- hydrogenases. Recently, the three-dimensional structures of the Fe-hydrogenases from Clostridium pasteurianum [14] and Desulfovibrio desulfuricans [15] were published. They have a multidomain s tructure with numerous [Fe–S] clusters [16] including a novel type of [Fe–S] cluster (H-cluster) within the catalytic site. This H-cluster consists of a conventional [4Fe)4S] c luster bridged by the sulfur atom of a cysteine residue to a unique binuclear iron subcluster [17]. Fe-hydrogenases from green algae m ediate a light driven hydrogen evolution after an anaerobic adaptation [ 4], but this H 2 -production does not occur under photosynthetic O 2 -evolving conditions [18,19]. The electrons can be supplied b y m etabolic oxidation of organic compounds with the release of carbon d ioxide [20,21]. This light dependent electron transport is 3-(3,4-dichlorophenyl)-1,1- dimethylurea (D CMU)-insensitive and requires only pho- tosystem I activity [22]. The role of the hydrogenase in green algae growing under photosynthetic co nditions in the natural environment has been unclear for a long time. Recently it was shown that s ulfur d eprivation in C. rein- hardtii cultures caused anaerobic conditions and, as a consequence, hydrogen production [23,24]. Under an anaerobic atmosphere, the hydrogen metabolism is t he only pathway for the algae to create enough ATP, which is required for the survival under this s tress condition [25]. Correspondence to T. Happe, Botanisches Institut der Universita ¨ t Bonn, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany Fax: + 49 228 731697, E-mail: t.happe@uni-bonn.de Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzochinon; SSH, suppression subtractive hybridization; TAP, Tris a cetate p hosphate; DIG, digoxygenin. Definitions: PS indicates photosystem I and II includ ing the reaction centers P 700 and P 680 ; Q and Z are the prim ary electron acceptors of the PS II or PS I, respectively; PQ refers to the plastoquinone pool. Note: the authors would like to d edicate this paper to Herbert Bo ¨ hme, who has retired because of a malignant disease. Note: the nucleotide sequ ence re port ed in this paper has be en submitted to the Gen Bank/EB I Data Bank w ith accession number CRE012098. (Received 18 June 2 001, revised 17 December 2001, accepted 18 December 2001) Eur. J. Biochem. 269, 1022–1032 (2002) Ó FEBS 2002 The Fe-hydrogenase of C. reinhardtii was purified to homogeneity and biochemically characterized [4]. The monomeric enzyme of 48 kDa is only synthesized after anaerobic adaptation and is located in the chloroplast stroma [26]. Despite the great interest in b iological H 2 evolution i n green algae, all attempts to isolate the hydrogenase gene from C. reinhardtii h ave so far not been successful. With the suppression subtractive hybridization (SSH) technique, a DNA fragment was isolated that showed similarity to Fe-hydrogenases. The full-length cDNA clone encoding HydA was obtained by screening a kgt11 expression library. This gene bank was constructed with poly(A) + RNA from anaerobically adapted C. reinhardtii cells. T he differ- ential regulation of protein biosynthesis dur ing a naerobic adaptation is discussed based on Northern blot analysis. The results present fundamental data for studying the hydrogen metabolism in photosynthetic eukaryotes. On the b asis of this research, we have r ecently published the isolation and characterization of the hydA gene from the green alga Scenedesmus obliquus [27]. MATERIALS AND METHODS Algae strains, culture conditions and anaerobic adaptation Wild-type Chlamydomonas reinhardtii 137c(mt+) strain was originally obtained from the Chlamydomonas Culture Collection at Duke University. The strain was grown photoheterotrophically [28] in batch cultures at 25 °C under a continuous irradiance of 150 lmol photonsÆ(m 2 Æs) )1 . Cultures containing TAP ( Tris acetate phosphate) m edium were flushed vigorously with air containing 5% CO 2 . Cells were harvested by centrifugation (8 min, 5000 g) in the mid- exponential growth stage (1–2 · 10 6 cellsÆmL )1 ). The pellet was resuspended in 0.02 vol. of fresh TAP medium and th e algae were a naerobically adapted b y flushing the s olution withargoninthedark. Hydrogen evolution assay Hydrogenase activity of C. reinhardtii was determined in vitro with reduced methyl viologen using a gas chro- matograph (Hewlett Packard 5890 A Series II, column: molecular Sieve 5 A ˚ , Mesh 60/80). The assay, containing in a final volume of 2 mL Pipes pH 6.8 (20 m M ), Na 2 S 2 O 4 (20 m M ), methyl viologen (5 m M ), was incubated a naero- bically at 25 °C for 20 min. One unit is defined as the amount of hydrogenase evolving 1 lmol H 2 Æmin )1 . Purification of the Fe-hydrogenase and amino-acid sequence Cells from a 40-L culture of C. reinhardtii were harvested by ultra fi ltration through an Amicon U ltrafiltration S ystem DC 10 LA, equipped with a hollow-fiber filter. The pellet was resuspended in 200 mL TAP medium. After anaero bic adaptation by flushing the solution with argon for 1 h in th e dark, all steps were performed under strictly anaerobic conditions [4]. The isolated Fe-hydrogenase was chemically cleaved by cyanogen bromide (CNBr). After separation of the CNBr f ragments on an SDS polyacrylamide gel, f our peptides were blotted onto a poly(vinylidene d ifluoride) membrane and were sequenced. Automated Edman degra- dation was performed with an Applied B iosystem model 477 A sequencer with online analysator model 120 A. RNA blot hybridization Total nucleic acids were isolated from algae grown under aerobic conditions and after anaerobic adaptation accord- ing to Johanningmeier & Howell [29]. Poly(A) + RNA was isolated using the RNA Kit (Qiagen); 10 lgtotalRNAor 0.5 lgpoly(A) + RNA were separated on each lane of 1.2% agarose gels in formaldehyde [30]. The RNA was trans- ferred to nylon membranes (Hybond + , Amersham) and hybridized with RNA probes, which were labeled with digoxygenin (DIG)-dUT P by in vitro transcription. Tran- scripts of the hydA gene were detected using a 1.0-kb SmaI cDNA fragment (Fig. 1). A DIG-dUTP labeled cDNA, which encodes the malate dehydrogenase, was used as control for a constitutive expressed gene. Suppression subtractive hybridization (SSH) SSH was performed with the Clontech PCR-select TM cDNA Subtraction Kit (Clontech Lab oratories Enc., Palo Alto, CA, USA) according to t he manufacturer’s recom- mendations, except for modifications of the PCR and hybridization conditions. The mRNA was isolated from aerobically grown cells (driver) and from anaerobically adapted algae ( tester). The driver and t ester cDNAs w ere denatured separately for the first hybridization at 100 °Cfor 30 s and then incubated for 10 h at 68 °C. For the second hybridization, driver cDNA was denatured at 100 °Cfor 30 s, then directly added to the pooled mix of the previous hybridization, and incubated at 68 °C for 20 h. Primary and secondary P CR con ditions were altered to increase the specificity o f t he amplification. The P CR conditions with subtracted cDNA were as follows: 25 cycles each 94 °Cfor 30 s, 68 °C for 30 s, and 72 °C for 1 min. The subtracted cDNA was s ubjected to a second round of nested PCR, Fig. 1. Sc hematic map of the cDNA and the genomic DNA region of hydA from C. reinhardtii. (A) Structural features of the hydA cDNA. Coding regions are marked as large arrows with the transit peptide shown in black. Lines indicate 5¢ and 3¢ URTs. (B) The mosaic structure of hydA is illustrated by gray (exons) and white boxes (introns). The RNA and DNA probes that were used for blotting experiments are no ted. Ó FEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1023 using the same PCR conditions with a decreased number of 15 cycles. Specific primers were used for the identifi cation of the amplified hydA cDNA fragment. From the N-terminal amino-acid sequence a degenerate oligonucleo- tide Hyd5 [5¢-GCCGCCCC(GC )GC(GCT)GC(GCT)GA (AG)GC-3¢] was synthesized, t aking into account known C. reinhardtii amino-acid sequences. The second primer Hyd2 (5¢-CCAACCAGGGCAGCAGCTGGTGAA-3¢) was deduced from the conservative amino-acid sequence motif of Fe-hydrogenases FTNaCl/CitPC. PCR was performed using either Hyd5 or Hyd2 and the nested PCR primer 2R from the Clontech Subtraction Kit. The PCR conditions were as follows: 20 pmolÆmL )1 of each primer were used; 35 cycles (denaturing at 95 °Cfor40s, annealing at 54 °C for 1 min, and extension at 72 °Cfor 1 m in). The amplified cDNA fragments were cloned i nto the T overhang vector pGEMÒ-T Easy (Promega). Screening of the cDNA library, cloning and sequencing A cDNA library was constructed u sing the Stratagene ZAP Express cDNA synthesis Kit (Stratagene, La Jolla, CA, USA) with 5 lg mRNA of anaerobically adapted cells of C. reinhardtii. Double-stranded cDNA was ligated into the ZAP Express vector, packaged with the Gigapack Gold Kit, and transfected into Escherichia coli XL Blue MRF – cells. The primary recombinant library con tained 5 · 10 6 recombinant phages and was amplified according to the manufacturer’s instructions. A 366-bp PCR fragment was radiolabeled with [a- 32 P]dCTP using the random-primer m ethod [31]. Approximately 5 · 10 5 plaques were analyzed under strin- gent hybridization conditions, resulting in 20 positive signals. The pBK-CMV phagemid vector with the different cDNAs was excised and used as a template for PCR, which was performed by using Hyd2 and Hyd5 primers at an annealing temperature of 56 °C for 1 min. Four plasmids contained cDNA fragments that showed similarities to the 366-bp fragment. All cDNA fragments were partially sequenced, and the largest clone pAK60 was completely sequenced. Sequencing was carried out by the dideoxy nucleotide triphosphate chain-termination method using the T 7 sequencing Kit (Pharmacia Biotech). Both strands of genomic and cDNA of hydA were completely sequenced using a nested set o f unidirectional deletions [32] or hydA specific synthetic oligonucleotides. The sequences of the Fe-hydrogenase are available under accession number CRE012098. Primer extension experiments were performed as described previously [27] using a 22-mer oligonucleotide (5¢-AATAGGTGGTGCGATGAAGGAG-3¢), which is complementary to the 5¢ end o f the hydA transcript. Expression studies in E. coli and Western blot analysis The coding region of hydA was amplified by PCR. The primers were identical to the cDNA sequences coding for the N- and the C-terminus of the mature protein plus several additional bases including NdeIandBamHI restric- tion sites, respectively (underlined). The oligonucleotide sequences were: HydNde (5¢- CATATGGCCGCACCCG CTGCGGAGGCGCCT-3¢), HydBam (5¢-CC GGATCC TCAAGCCTCTGGCGCTCCTCA-3¢). The hydA gene, corresponding to amino acids 57–497, was amplified, confi rmed by sequences analysis and cloned into corresponding sites of the pET9a e xpression v ecto r (Pro- mega). The constructed p lasmid was then t ransformed into E. coli strain BL21(DE3). After induction with 1 m M isopropyl-thio-b- D -galactoside, the cells were resuspended in lysis buffer. Crude extracts from C. reinhardtii were isolated by harvesting cells after indicated anaerobic adapta- tion times. The p ellet was resuspended in s olubilization buffer and incubated w ith vigorous vortexing at RT for 30 min. The protein extracts from C. reinhardtii and E. coli were separat - ed by 12% SDS/PAGE and blotted onto a poly(vinylidene difluoride) membrane. Affinity-purified antibodies were diluted 1 : 200 and used for Western blot analyses [26]. Sequence analysis and protein modeling Nucleic acid and protein s equences were analyzed with the programs SCI ED CENTRAL (Scientific Educational Software, Durham, NC, USA) and CLUSTALW [33]. The BLAST server [34] of the National Center for Biotechnology Information (Bethseda, MD, USA) was used for database searches. RESULTS Isolation of cDNA clones, which are differentially expressed during anaerobic adaptation In order to a mplify a p art of the hydrogenase gene in a PCR reaction, degenerate oligonucleotides corresponding to conserved regions of known Fe-hydrogenases were used. All products of expected sizes were cloned and sequenced, but they showed no homologies to other hydrogenases (data not shown). Examinations were then focused on the process of anaerobic adaptation in C. reinhardtii, because the Fe-hydrogenase was only detected under these conditions [26]. Therefore, we isolated two different populations of mRNA and took advantage of the SSH technique [35]. Poly(A) + RNA was isolated from aerobically grown C. reinhardtii cells and from a cell suspension flushed 15 min with argon. After cDNA synthesis, subtractive hybridization, and PCR experiments (see Material and methods), the amplified PCR f ragments were cloned and sequenced. Twenty different clones containing inserts of 184–438 bp were analyzed (Table 1). In transcription ana- lyses, 15 of them showed an increased signal under anaerobic conditions (data not shown). Database comparisons (using GenBank/EBI DataBank) confirmed that eight of these cDNA fragments are similar to genes encoding proteins of the cytoplasmic ribosome complex. The sequences of six clones did not co rrespond to any entries in the databases. Four of these novel clones showed differences in expression between aerobically grown and anaerobically adapted c ul- tures. Another cDNA fragment (No. 7) indicated similarity to the 5¢ region of the Fe-hydrogenase from bacteria. Analysis of the hydA cDNA and genomic sequences A kgt11 cDNA expression library was c onstructed u sing poly(A) + RNA from anaerobically adapted cells (15 min). Two oligonucleotides were generated on the basis of the cDNA fragment isolated by SSH and the N-terminal sequences of the purified hydrogenase. They were used to 1024 T. Happe and A. Kaminski (Eur. J. Biochem. 269) Ó FEBS 2002 amplify a 366-bp cDNA fragment that showed 41% identity to the corresponding part of the Fe-hydrogenase of C. pasteurianum. The fragment was labeled with [a- 32 P]dCTP an d u sed t o s creen the cDNA library. Four independent cDNA clones with different sizes of 2.4-, 1.9-, 1.7- and 1.6-kb were identified and sequenced. The nucleo- tide sequence of the largest clone, 2399-bp, revealed an ORF encoding a polypeptide of 497 amino a cids (Fig. 1). The cDNA also contained a 5¢ UTR (158-bp) and a longer 3¢ UTR ( 747-bp e xcluding the polyadenylated tail). Charac- teristic features of other C. reinhardtii cDNA clones, e.g. a high average G/C content (62.1%) and a putative polyade- nylation signal (TGTAA, 727-bp downstream of the stop codon [ 36]) were found. The transcription start position was confirmed by primer extension 158-bp upstream of the ATG start codon (Fig. 2). Approximately 5-kb of the hydA genomic region was determined. The coding sequence is interrupted by seven introns (Fig. 1) with sequences at their 5¢ and 3¢ ends corresponding to the typical splicing sequences from eukaryotes [37]. T he promoter region does n ot contain a putative TATA box or any other known transcription motifs. The sequence data were submitted to the GenBank/ EBI DataBank under accession number CRE012098 three years ago. M eanwhile parts of the cDNA sequen ce were determined by another group and deposited under accession number AF289201. Southern hybridization experiments were perfor med at high stringency using a PCR fragment as p robe (Fig. 3). They showed the presence of one hybridizing signal of similar intensity in different digestions, suggesting that HydA is encoded by a single copy gene in the C. reinhardtii genome. The same hybridization pattern was observed even under low stringency conditions (hybridization temperature 50 °C; data not shown). Characterization of the Fe-hydrogenase HydA The mature polypeptide consists of 441 amino acids with a calculated molecular mass of 47.5 kDa and a predicted isoelectric point of 5.6. The N-terminal 56 amino acids probably function as transit peptide, because they show characteristics of polypeptides that route proteins into the chloroplast stroma [38]. The stromal targeting domain i s probably cleaved by a stromal peptid ase at the conserved cleavage motive Val-Ala-Cys-Ala (Fig. 2). In addition to the detection of the protein using antibodies raised against the Fe-hydrogenase, t he localization o f t he mature protein in the chloroplast stroma is indicated by a high content of hydroxylated and basic amino acids in the t ransit peptide sequence [39]. The deduced amino-acid sequence of the mature HydA polypeptide from C. reinhardtii shows 60% identity and 71% similarity to the Fe-hydrogenase o f S. obliquus [27], which was recently isolated on the basis of the data of this work. Comparisons with NiFe-hydrogenases o f bacteria (including the photosynthetic cyanobacteria) had obviously lower scores, e.g. 25% similarity with the NiFe-hydrogenase (HoxH) of Ralstonia eutropha [1]. A conserved d omain of about 300 amino acids is found in the C-terminal part of all Fe-hydrogenases. The sequences are highly conserved, especially in the region that is involved in the c atalytic mechanism (H-cluster), indicating structural similarity between Fe-hydrogenases [14]. Four cysteine residues at positions 114, 169, 361 and 365 might coordinate the H-cluster in C. reinhardtii. Twelve strictly conserved amino a cids of HydA proteins p robably define a binding pocket surrounding the a ctive center as shown by structural data of C. pasteurianum and D. desulfuricans [14,15]. All of them are present in the C. reinhardtii protein (Pro37, Ala38, Thr74, Ala78, Cys113, Pro138, Met167, Lys 172, Glu175, Phe234, Val240 and Met359; Fig. 4). An interesting inser- tion of 45 amino acids was only identified at the C-terminus of the C. reinhardtii polypeptide (position 285–329). The N-terminal region of the green algae protein is much shorter and completely different to all known Fe-hydrog- enases. Amino-acid sequence analyses have indicated t hat Fe-hydrogenases i n general contain two [4Fe)4S] clusters (F-cluster) in a ferredoxin-like domain. They might be involved in the transfer of electrons from the donor to the catalytic center [15]. This N-terminal domain with the F-cluster o r o ther conserved cysteines is completely mi ss- ing i n HydA o f C. reinhardtii. A novel electron transport pathway i s postulated f rom the exogenous donor (ferred- oxin) directly to the H-cluster. Protein sequencing of the enzyme and recombinant expression of HydA in E. coli To verify that the hydA ORF encodes t he Fe-hydrogenase of C. reinhardtii, the enzyme was purified according to Happe & Naber [4]. The purified protein was able to evolve Table 1. Summary of anaerobically induced c DNA clones generated from Chlamydomonas reinhardtii by suppression subtractive hybridiza- tion (SSH). –, n ovel s equence . +, on ly or stronger expression in anaerobically grown cells. No. Size (bp) a Gene b mRNA (kb) c Differential expression d 1 281 Ribosomal protein S8 0.8 + 2 312 – 2.4 + 3 192 – 1.8 – 4 369 Ribosomal protein L17 1.2 + 5 301 Catalase 2.1 – 6 297 – 1.6 + 7 232 Fe-hydrogenase 2.4 + 8 317 Ribosomal protein S8 0.8 + 9 184 Malate-dehydrogenase 1.8 – 10 412 Ribosomal protein S15 0.7 + 11 309 – 2.2 + 12 243 Ribosomal protein L12 0.9 – 13 321 – 1.1 + 14 272 Ribosomal protein S8 0.8 + 15 251 Ribosomal protein L37 0.7 – 16 380 14-3-3 protein 1.5 + 17 438 Enolase 2.0 + 18 384 Aldolase 1.7 + 19 273 Ribosomal protein S18 0.8 + 20 195 – 1.9 + a Size of PCR-generated inserts that were determined after sequencing. b Sequence identities based on comparison with General Bank/EMBL database. c Estimation of the size (kb) of mRNA by Northern analysis. d Relative expression levels are based on Northern analysis with poly(A) + RNA. Ó FEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1025 hydrogen, w hen incubated with reduced methyl viologen. After proteolytic digestion with cyanogen bromide, four bands of 4, 8, 9 and 11 kDa were detected after SDS/PAGE separation (data not shown). Two fragments (9 and 11 kDa) were sequenced by Edman degradation. They are identical with the deduced amino-acid sequence of hydA (sequences are shadowed in gray in Fig. 2). The fragment corresponding to the cDNA region between 158 and 1636 bp of hydA was NdeI–BamHI cloned into the expres- sion vector pET9a. The heterologous expressed protein was Fig. 2. Nuc leotide sequence of the hydA cDNA and the deduced amino-acid sequence of the hydrogenase from C. reinhardtii. The sequence was submitted to the GenBank/EBI Data- Bank under accession number CRE012098. An arrow marks the transcription start point. The ATG start codon and the TGA stop codonaredrawninboxes.Boldfaceletters indicate the cDNA sequence. Gray shadows mark amino acids corresponding to polype p- tide sequence s that wer e determin ed by sequencing the N-termin us of the protein. Black shadows mark the putative transit peptide, and the unde rlined amino acids ind i- cate the putative cleavage site for the endo- peptidase. Boldface double underlined letters indicate a signal for polyadenylation. 1026 T. Happe and A. Kaminski (Eur. J. Biochem. 269) Ó FEBS 2002 detected using antibodies raised against the Fe-hydrogenase (Fig. 5). Both the purified Fe-hydrogenase of C. reinhardtii and the overexpressed enzyme had the same size ( 47.5 kDa). No hydrogenase activity could be measured within the lysate of the induced E. coli cells. This result is in agreement with S tokkermans et al. [40] and Voordouw et al. [41] who also detected no H 2 -production of the recombinant expressed Fe-hydrogenase from Desulfovibrio vulgaris in E. coli cells. An explanation might be the inability of E. coli to assemble the unique active site of the Fe-hydrogenases. It is known that E. coli has only three NiFe-hydrogenases with a different maturation s ystem for the catalytic center [42]. Induction of gene expression during anaerobic adaptation In aerobically grown cells, neither hydrogenase activity [4] nor protein can be identified by immunoblot analysis. However, HydA can be detected only 15 min after anaer- obic adaptation (Fig. 6). The expression of the hydA gene is probably regulated at the transcriptional level. Total RNA was isolated from cells that had been anaerobically adapted by flushing with argon for 0, 15, and 30 min. Northern blot hybridization demon- strated that the hydA gene is expressed very rapidly after the beginning of anaerobic adaptation. No transcript could be detected before adaptation (t ¼ 0), but a significant signal occurred after just 15 m in of anaerobiosis (Fig. 6). The size of the transcript (2.4 kb) confirmed t he full-length of the isolated hydA cDNA fragment. DISCUSSION Differentially expressed genes during anaerobic adaptation Hydrogen metabolism induced b y anaerobic conditions is well es tablished in g reen algae. I n the absence of oxygen, C. reinhardtii, a nd also plants, s witch their metabolism t o fermentation [43,44]. In the light, algae degrade cellular starch via g lycolysis [45 ] and hydrog en gas is evolved. It has been suggested that reducing equivalents from the glycolysis or the c itric acid c ycle can t ransfer t heir electrons to the photosynthetic electron transport chain [46]. However, the molecular principles of the gene induction under anaerobic conditions in C. reinhardtii are poorly understood. In this present work, we investigated the patterns of gene expression in aerobically grown and anaerobically adapted cells by isolating differentially expressed genes. The SSH method combines subtractive hybridization with PCR [47] to generate a population of PCR fragments enriched with gene sequences that are only expressed under anaerobic conditions. Compared to other PCR-based cloning strate- gies, such as differential display [48], the great a dvantage of SSH is that fewer false positives are generated; 70% of the cloned fragments represented differentially expressed genes. Among the 20 sequenced cDNA clones, we found three DNA fragments encoding the ribosomal S8 protein. Most of the other sequences (eight of 20) also corresponded to ribosomal protein sequences. This might indicate that the transcripts of the ribosomal protein genes (rps, rpl) accumulate under stress conditions. This is in good agree- ment with Dumont et al. [49] who found that an accumu- lation of ribosomal p roteins takes place un der phosphate starvation. Moreover, two of the identified cDNAs enco de for proteins (aldolase, enolase), which are induced in other organisms by anaerobic stress [50,51]. Anaerobic treatment of maize seedlings alters the profile of total protein synthesis [52,53]. It is known that the induction of the anaerobic proteins is the result of a n increased mRNA level. Maize (Zea mays L.) responds to anaerobic stress by redirect- ing t he synthetic machinery towards the synthesis of s ome enzymes involved in glycolysis or sugar-phosphate metabolism [54]. HydA belongs to a new class of Fe-hydrogenases HydA of C. reinhardtii, the first isolated gene encoding a hydrogenase of a photosynthetic eukaryotic cell, repre- sents a novel type of Fe-hydrogenases. Parts of the deduced amino-acid sequence of the cDNA correspond to the polypeptide sequence of the tryptic fragment (VPAPGSKFEELLKHRAAARA), and the N-terminus Fig. 3. Southern hybridization analysis of hydASouthern blot analysis. C. reinhardtii genomic DNA was digested with three different restric- tion endonucleases (SacI, HincII, PstI) a nd 1 0 lg of DNA was loaded per lane. The DIG-dUTP labeled DNA fragment (750-bp) was used for hybridization as indicated in Fig. 1. Ó FEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1027 Fig. 4. Mult iple sequence alignments of Fe-hydrogenases. Sequence comparison o f the deduced amino-acid se quence of HydA from C. reinhardtii with Fe-hydrogenases published for other organisms. The protein a lignment was done by using the Vector NTI p rogram ( INFORMAX ). Black highlighted letters indicate amino acids identical to th e HydA protein. Gray shadowed amino acids indicate conserved changes o f the amino acids. The abbreviations of the organisms are: C. r., Chlamydomonas reinhardtii (CRE012098); S. o., Scenedesmus obliquus [27]; M. e., Megasphaera elsdenii [11]; D. d., Desulfovibrio desulfuricans [15]; C. p., Clostridium p asteuria num [12]; N. o., Nyctotherus ovalis [10]. Black arrows indicate conserved cyste ines, g ray one s t he residu es tha t are n ec essary for the formation of the H -clu ster an d white a rrows re fer to the conserved cysteine s of the F-cluster, which is lacking in C. reinhardtii and S. obliquus. 1028 T. Happe and A. Kaminski (Eur. J. Biochem. 269) Ó FEBS 2002 (AAPAAAEAPLSHVQQALAELAKPKD) f rom t he purified native enzyme [4]. Further evidence that the isolated cDNA encodes an Fe-hydrogenase is the fact that the recombinant HydA s pecifically r eacts with the antibodies raised against the active enzyme. The amino-acid sequence of HydA shows only c onsiderable similarity to Fe-hydro- genases but not to NiFe-hydrogenases. The Fe-hydrogenase family is one c lass of hydrogenases defined by Vignais et al. [55]. The enzymes have been identified in a small group of anaerobic microbes, where they often catalyze the reduction of pr otons with a high specific activity to yield hydrogen [16]. Interestingly, Fe-hydrogenases were not found in cyanobacteria, the free-living ancestor of plastids, suggest- ing a noncyanobacterial origin for the algal hydrogenases. The important structural features found among the amino-acid sequences of Fe-hydrogenases are also present in the C. reinhardtii hydrogenase sequence. A highly conserved domain of about 130 amino acids was detected in the C-terminal part of the protein. The designated active- site domain [14] consists of an atypical [Fe–S] cluster (H-cluster). In C. pasteurianum, the H -cluster contains six Fe atoms a rranged as a [4Fe)4S] subcluster br idged to a [2Fe] s ubcluster by a s ingle cysteinyl sulfur. T he [4Fe 4S] subcluster is coordinated to the protein by four cysteine ligands, which have also been found in the amino-acid sequence of C. reinhardtii (Fig. 4). A number of mostly hydrophobic amino-acid residues define the environment of the active site and might have a function in protecting the H-cluster from solvent access [14]. In contrast to all Fe-hydrogenases including HydA of S. obliquus,the enzyme of C. reinhardtii has an interesting additional protein domain. A small insertion of 45 amino acids between residue Ser284 and Val330 builds an external loop of the protein that might b e involved in electrostatic binding of the natural electron donor ferredoxin (M. Winkler, B. Neil & T. Happe, unpublished results). In the N-terminus of other Fe-hydrogenases further cysteine residues w ere found that bind accessory iron sulfur clusters. A ferredoxin homologous domain (F-cluster) coor- dinates two [4Fe)4S] clusters in all non algal Fe-hydrogen- ases [3,11]. An additional [4Fe)4S] cluster and one [2Fe)2S] center were de tected within the Fe-hydrogenases of C. paste- urianum [14]. Based on similarities of the primary sequences, the same cofactors are proposed for Thermotoga maritima and Nyctotherus ovalis [10,13]. The F-cluster is responsible for the electron transfer from t he electron donor (mostly ferredoxin) to t he active center [56]. It has been suggested that the proteins containing two [4Fe–4S] clusters a re ancestors of the Fe-hydrogenases [55]. The N-terminus of the C. reinhardtii and S. obliquus proteins is strongly reduced, and conserved cysteines were also not found. Therefore we suggest that all accessory [Fe–S] clusters are m issing in the a lgal hydrogenases. The native protein of C. reinhardtii is located in the chloro plast stroma [26]. T he first 56 amino acids of the u n processed enzyme probably function as a transit peptide, because they were not characterized i n t he purified hydrogenase and a putative peptidase cleavage site (Val-Ala-Cys-Ala) [38,39] could be detected at the end of this fragment. The natural electron donor of the hydrogenase in C. reinhardtii is the ferredoxin (PetF) of the photosynthetic electron transport pathway [26]. Measuring the H 2 -evolution, we have shown that the hydrogenase activity is directly linked to the 47.5-kDa subunit [4]. As w e have not found a second subunit necessary for hydrogenase activity, we suggest that a direct electron tran sfer from PetF to H ydA takes place. In vitro, a hydrogen evolution by HydA was only measured with plant-type [2Fe)2S] ferredoxins such as PetF of C. reinhardtii, S. obliquus and s pinach as electron mediators (data not shown). Fig. 5. Overexpression of hydA in E. coli. The hydA gene corre- sponding to amino acid 57 to residue 497 was cloned NdeI–BamHI into th e pET9a vector. The HydA p rotein was overexpressed upon induction with i sopropyl thio- b- D -galactoside . L anes 1, purified hydrogenase from C. reinhardtii; Lanes 2 , protein extract from 2 -h induced E. c oli cells was separated on an SDS polyacrylamide gel. The molecularmassmarker(Bio-Rad)indicatesrelativemolecularmasses in kDa. (A) The SDS polyacrylamide gel was stained w ith Coomassie Blue. (B) Western blo tting and imm unodete ction was carrie d out a s described previously [24]. Fig. 6. D ifferential expression of the hydA gene shown by Northern blot analysis. The aerobically grown C. reinhardtii cells were c entrif uged, resuspended in buffer and anaerobically adapted by flushing the solution with argon. Adapted cells were harvested at 0, 15, 30 and 60 min, and RNA of the cells and proteins were isolated a s d escribed in Materials and methods. (A) Northern hybridization with the hydA specific probe. (B) Hybridization with a constitutively expressed gene (malate dehydrogenase). RNA size standards (Roche) in kb are indi- cated on t he le ft. (C) Immunoblot w ith a ffinity-purified antibodies on the right. Ó FEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1029 Why do the photosynthetic green algae still keep the anaerobically induced hydrogenases? The most likely explanation is that the enzymes ensure t he survival of the cells under these anaerobic conditions. Melis et al. have shown that H 2 -evolution is the only mechanism available to the algae for generating sufficient amounts of ATP under S-depleted anaerobic conditions [23,24]. It is known that C. reinhardtii is still able to photoproduce hydrogen when photosystem II is inhibited by DCMU, but no H 2 -evolu- tion occurs after an addition of 2,5-dibromo-3-methyl- 6-isopropyl-p-benzochinon (DBMIB; F ig. 7) [27]. Under anaerobic conditions, accumulated reducing equivalents from the fermentative metabolism c annot be oxidized via respiration, as the electron acceptor o xygen is missing. The NAD(P)H reductase protein complex has r ecently been isolated from plants [58], a nd inhibitor e xperiments have shown evidence o f a membrane-bound, chloroplast-located reductase in C. reinhardtii [59]. The light-dependent electron transport of the H 2 -evolution is driven by p lastoquinone and photosystem I. The donor ferredoxin transfers electrons in a last step to the hydrogenase and molecular hydrogen is released (Fig. 7). Regulation of hydA at the transcriptional level Our studies have shown that there is a correlation between the increase of hydrogen production and the anaerobic adaptation, which was documented by activity measure- ments [ 26] and immunoblots (Fig. 6). It i s likely that t he induction of hydA is regulated on the level of transcription. We observed t hat the amount of mRNA increased directly with the measured H 2 -evolution. In C. reinhardtii, a dramatic change in the hydrogenase transcript level occurs during the shift from an aerobic to an anaerobic atmo- sphere, which means that the transcription is regulated by the oxygen status of the cells. A very rapid increase of the hydA transcript was detected in the first 3 0 min of anaerobiosis. This quick increase of gene transcription is only reported for the cyc6 gene in C. reinhardtii [60] and for the SAUR (Small Auxin-Up RNA) genes in plants [61]. Interestingly, the hydA gene of S. obliquus is constitutively transcribed under aerobic conditions [27] indicating another regulation system for the expression of the hydrogenase. At the m oment it is not clear if this effect rests upon a new synthesis or a higher stability o f the hydA mRNA. As with other nuclear genes, the promoter region of the hydA from C. reinhardtii contains no conserved TATA box or other motif similarities [62]. As no defined motif structures in the promoter region of hydA have been found, further g enetic analyses are necessary to investigate the rapid induction of hydA in C. reinhardtii. ACKNOWLEDGEMENTS The authors wish to t hank D. Nabe r for he lpful advic e and discussions. We thank Dr R. Deutzmann (Universita ¨ t Regensburg) for determ ina- tion of the amino-acid sequences. This work was supported by t he Deutsche Forschungsgemeinschaft (Ha 2555/1-1). REFERENCES 1. Friedrich, B. & Schwartz, E. (1993) M olecular biology of hydro- gen utilization in aerobic chemolithotrophs. Annu. Rev. Microbiol. 47, 351–383. 2. Przybyla,A.E.,Robbins,J.,Menon,N.&PeckJr,H.D.(1992) Structure–function relationships of nickel-containing hydrogen- ases. FEMS Microbiol. Rev. 88, 109–136. 3. Bui, E.T. & Johnson, P.J. (1996) Identification and characteriza- tion of Fe-hydrogenases in the hydrogenosome of Trichomonas vaginalis. Mol. Biochem. Parasitol. 76, 305–310. 4. Happe, T. & Naber, J.D. (1993) Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur. J. Biochem. 214, 475–481. 5. Albracht, S.P.J. (1994) Nickel hydrogenases: in search of the active site. Biochim. Biophys. Acta 1188, 167–204. 6. Volbeda, A., C haron, M H., Piras, C., Hatchikian, E.C., F rey, M. & Fontecilla-Camps, J.C. (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580–587. Fig. 7. Sc heme of the light-dependent photoevolution of hydrogen i n green algae. The electron s for hydrogen evolution are fed into th e pho tosynth etic electron transport chain either via PS II or via the plastoquinone pool after oxidation of reducing equivalents. The natural electron donor PetF transfers the electrons f rom PS I to the hydrogenase. PS i ndicates the photosystem I and II incl uding t he react ion centers P 700 and P 680 ;QandZare the primary electron acceptors of the PS II or PS I, respectively; PQ shows the plastoquinone pool, Cyt the cytochromes, PC plastocyanin, Fd ferredoxin, FNR the ferredoxin N ADP reductase, Red the NAD(P)H reductase, and H 2 ase the hydro genase. DCMU, 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-bezochinon. 1030 T. Happe and A. Kaminski (Eur. J. Biochem. 269) Ó FEBS 2002 7. Adams, M.W.W. (1990) The structure and mechanism of iron- hydrogenases. Biochim. Biophys. Acta 1020, 115–145. 8. Berkessel, A. & Thauer, R.K. (1995) On the mechanism of catalysis by a metal-free hydrogenase from methanogenic archaea: enzymic transformation of H 2 without a metal. Angew. Chem. Internat 34, 2247–2250. 9. Zirngibl, C., van Do ngen, W ., Sc hwo ¨ rer, B., v on Bu ¨ nau, R., Richter, M., Klein, A. & Thauer, R.K. (1992) H 2 -forming meth- ylenetetrahydromethan opterin dehydrogenase, a novel type of hydrogenase without iron sulfur cluster in methanogenic archaea. Eur. J. Biochem. 208, 511–520. 10. Akhmanova, A., Voncken, F., van Alen, T., van Hoek, A., Boxma, B., Vogels, G., Veenhuis, M. & Hackstein, J.H. (1998) A hydrogenoso me with a gen ome. Nature 396, 527–528. 11. Atta, M. & Meyer, J. (2000) Ch aracterization of the gene encoding the [ Fe]-hydrogenase f rom Megasphaera elsdenii. Biochim. Bio- phys. Acta 1476, 368–371. 12. Meyer, J. & Gagnon, J. (1991) Primary structure of h ydrogenase I from Clostridium pas teurianu m. Biochemist ry 30, 9697–9704. 13. Verhagen, M F., O’Rourke, T. & Adams, M.W.W. (1999) The hyperthermophilic bacterium, Thermotoga maritima,containsan unusually complex iron-hydrogenase: amino acid sequence ana- lyses versus biochemical characterization. Biochim. Biophys. Acta 1412, 212–229. 14. Peters, J.W., Lanzilot ta, W.N., L emon, B.J. & S eefeld t, L.C. (1998) X-ray crystal structure of the Fe-only h ydrogenase (Cpl) from Clostridium pasteurianum to 1.8 A ˚ ngstom resolution. Science 282, 1853–1858. 15. Nicolet, Y ., Piras, C., Legrand, P., Hatchikian, C.E. & Fontecilla- Camps, J.C. (1999) Desulfovibrio desulfuricans iron hydrogenase: the s tructure shows unusu al coordination to an active site Fe binuclear c enter. Structure 7, 13–23. 16. Peters, J.W. (1999) Structure and mechanism of iron-only hydrogenases. Curr. Opin. Struct. Biol. 9, 670–676. 17. Adams, M.W.W. & Stiefel, E.I. (2000) Organometallic iron: the key to biological hydrogen metabolism. Curr. Opin. Chem. Biol. 4, 214–220. 18. Ben-Amotz, A. & Gibbs, M. (1975) H 2 metabolism in photosyn- thetic organisms. II. Light-dependent H 2 evolution by p reparation from Chlamydomonas, Scenedesmus and Spinach. Biochem. Bio- phys. Res. Commun. 64 , 355–359. 19. Biochenko, V.A. & Hoffmann, P. (1994) Photosynthetic hydrogen production in prokaryotes and eukaryotes: occurrence, mech- anism and function. Photosynthetica 30, 527–552. 20. Bamberger, E.S., King, D., Erbes, D.L. & Gibbs, M. (1982) H 2 and CO 2 evolution by anaerobically adapted Chlamydomonas reinhardtii F60. Plant Physiol. 69, 1268–1273. 21. Kessler, E . (1974) Hydrogenase, photoreduction and anaerobic growth. In Algal Phy siolog y and Biochemistry (Stewart, W.D.P., ed.) pp. 456–473 Blackwell Science, Oxford. 22. Stuart, T.S. & Gaffron, H. (1972) The mechanism of hydrogen photoproduction by several algae. The contribution of photo- system II. Planta 106, 101–112. 23. Melis, A., Zhang, L., Forestier, M., Ghirardi, M.L. & Seibert, M. (2000) Su stained photobiological hydro gen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol. 122, 127–135. 24. Zhang,L.,Happe,T.&Melis,A.(2001)Biochemicalandmor- phological c haracterization of sulfur-deprived and H 2 -producing Chlamydomonas reinhardtii (gr een a lga). Planta, in press. DOI 10.1007/s00425010066. 25. Melis, A. & Happe, T. (2001) Hydrogen production. Green algae as a source of energy. Plant Physiol. 127, 740–748. 26. Happe, T., Mosler, B. & Naber, J.D. (1994) Induction, localiza- tion and metal content of hydrogenase in Chlamydomonas rein- hardtii. Eur. J. Biochem. 222, 769–775. 27. Florin, L., Tsokoglou, A. & Happe, T. (2001) A novel type of iron hydrogenase in the green alga Scened esmus obliquus is linked to the photosynthetic electro n transport chain. J. Biol. Chem. 276, 6125– 6132. 28. German, D.S. & Levine, R.P. (1965) Cytochrome f and plasto- cyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc. Natl Acad. Sci. USA 54, 1665–1669. 29. Johanningmeier, U. & Howell, S.H. (1984) Regulation of light- harvesting chlorophyll-bi nding protein mRNA accumulation in Chlamydomonas reinhardtii. Possible in volvement of chlorophyll synthesis precursors. J. Biol. Chem. 259, 13541–13549. 30. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: a L aboratory M anual, 2 nd edn. Co ld Sp ring H arbor Laboratory Press, Cold Spring Harbor, NY. 31. Feinberg, A.P. & Vogelstein, B. (1983 ) A technique f or radio- labeling DNA restrict ion endonuc lease fragments to high specific activity. Anal. Biochem. 137, 266–267. 32. Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359. 33. Thompson, J.D., H iggins, D.G. & Gibson, T.J. (1994) CLUS TAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap pen- alties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. 34. Altschul, S.F., Madden, T.L., Scha ¨ ffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1985) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. 35. Diatchenko, L., Lau, Y.F., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D. & Siebert, P.D. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl Acad.Sci.USA93, 6025–6030. 36. Silflow, C.D ., Chisholm, R.L ., Conner, T.W. & Ranum, L .P. (1985) The two alpha-tubulin genes of Chlamydomonas reinhardtii code for slightly different proteins. Mol. Cell Biol. 5, 2389–2398. 37. Breathnach, R. & Chambon, P. (1981) Organization and expres- sion of eucaryotic split gen es c od ing f o r prote ins. Annu . R ev. Biochem. 50, 349–383. 38. Franzen, L.G., Rochaix, J.D. & v on H eijne, G. (1990) Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences. FEBS Lett. 260, 165–168. 39. Keegstra, K. (1989) Transport and routing o f proteins into chloroplasts. Cell 56, 247–253. 40. Stokkermans, J., van Dongen, W., Kaan, A., van den Berg, W. & Veeger, C. (1989) hyd gamma, a gene from Desulfovibrio vulgaris (Hildenborough) encodes a polypeptide homologous to the periplasmic hydrogenase. FEMS Microbiol. Lett. 49, 217–222. 41. Voordouw, G., Hagen, W.R., Kruse-Wolters, K.M., van Berkel- Arts, A. & Veeger, C. (1987) Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli. Eur. J. Biochem. 162, 31–36. 42. Rossmann, R., Maier, T., Lottspeich, F. & Bo ¨ ck, A. (1995) Characterization of a protease from Escherichia coli involved in hydrogenase maturation. Eur. J. Biochem. 227, 545–550. 43. Gfeller, R.P. & Gibbs, M . (1984) Fe rmentative metabolism of Chlamydomonas reinhardtii. I. Analysis of fermentative prod ucts from starch in dark and light. Plant Physiol. 75, 212–218. 44. Kennedy, R.A., Rumpho, M.E. & Fox, T.C. (1992) Anaerobic metabolism in plants. Plant Physiol. 100, 1–6. 45. Ohta, S., Miyamoto, K. & Miura, Y. (1987) Hydrogen evolution as a consumption mode of reducing equivalents in green alga fermentation. Plant Physiol. 83, 1022–1026. Ó FEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1031 [...]... for the photosynthetic membrane of Chlamydomonas reinhardtii Arch Microbiol 127, 245–252 60 Hill, K.L., Li, H.H., Singer, J & Merchant, S (1991) Isolation and structural characterization of the Chlamydomonas reinhardtii gene for cytochrome c6 Analysis of the kinetics and metal specificity of its copper-responsive expression J Biol Chem 266, 15060–15067 61 Li, Y. , Liu, Z.B., Shi, X., Hagen, G & Guilfoyle,... eukaryotic messenger RNA by means of the polymerase chain reaction Science 257, 967–971 49 Dumont, F., Joris, B., Gumusboga, A., Bruyninx, M & Loppes, R (1993) Isolation and characterization of cDNA sequences controlled by inorganic phosphate in Chlamydomonas reinhardtii Plant Sci 89, 55–67 50 Hake, S., Kelley, P.M & Freeling, M (1985) Coordinate induction of alcohol dehydrogenase 1, aldolase and other... aldolase and other anaerobic RNAs in maize J Biol Chem 260, 5050–5054 51 Lal, S.K., Elthon, T.E & Kelley, P.M (1994) Purification and differential expression of enolase from maize Plant Physiol 91, 587–592 52 Russell, D.A & Sachs, M.M (1992) Protein synthesis in maize during anaerobic and heat stress Plant Physiol 99, 615– 620 53 Sachs, M.M., Freeling, M & Okimoto, R (1980) The anaerobic proteins of maize Cell... X., Hagen, G & Guilfoyle, T.J (1994) An auxin-inducible element in soybean SAUR promoters Plant Physiol 106, 37–43 62 Silflow, C.D (1998) Organization of the nuclear genome In The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas (Rochaix, J.D., Goldschmidt-Clermont, M & Merchant, S., eds) pp 25–40, Kluwer Academic Publishers, Dordrecht, the Netherlands ... Kaminski (Eur J Biochem 269) 46 Gibbs, M., Gfeller, R.P & Chen, C (1986) Fermentative metabolism of Chlamydomonas reinhardtii III Photoassimilation of acetate Plant Physiol 82, 160–166 47 von Stein, O.D., Thies, W.G & Hofmann, M (1997) A high throughput screening for rarely transcribed differentially expressed genes Nucl Acids Res 25, 2598–2602 48 Liang, P & Pardee, A.B (1992) Differential display of. .. S.K., Lee, C & Sachs, M.M (1998) Differential regulation of enolase during anaerobiosis in maize Plant Physiol 118, 1285–1293 Ó FEBS 2002 55 Vignais, P.N., Billoud, B & Meyer, J (2001) Classification and phylogeny of hydrogenases FEMS Microbiol Rev 25, 455–501 56 Nicolet, Y. , Lemon, B.J., Fontecilla-Camps, J.C & Peters, J.W (2000) A novel FeS cluster in Fe-only hydrogenases Trends Biochem Sci 25, 138–142... 138–142 57 Moulis, J.-M & Davasse, V (1995) Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners Biochemistry 34, 16781–16788 58 Sazanov, L.A., Burrows, P.A & Nixon, P.J (1998) The plastid ndh genes code for an NADH-specific dehydrogenase: isolation of a complex I analogue from pea thylakoid membranes Proc Natl Acad Sci USA 95, 1319–1324 . Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii Thomas Happe and Annette Kaminski Botanisches. all Fe-hydrogenases including HydA of S. obliquus ,the enzyme of C. reinhardtii has an interesting additional protein domain. A small insertion of 45 amino