Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions Marc Forestier 1, *, Paul King 1 , Liping Zhang 1 , Matthew Posewitz 1 , Sarah Schwarzer 2 , Thomas Happe 2 , Maria L. Ghirardi 1 and Michael Seibert 1 1 National Renewable Energy Laboratory, Golden, CO USA; 2 Ruhr-Universitaet-Bochum, Lehrstuhl Biochemie der Pflanzen AG Photobiotechnologie, Bochum, Germany We have isolated and characterized a second [Fe]-hydro- genase gene from the green alga, Chlamydomonas reinhardtii. The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [Fe]-hydrogenases. We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O 2 , the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H 2 -gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe-S centers and the existence of extra sequences in the algal enzymes. To our knowledge, this work represents the first systematic study of expression of two algal [Fe]- hydrogenases in the same organism. Keywords: green algae; anaerobic induction; hydrogenase; sulfur deprivation; gene expression. Hydrogen metabolism, catalyzed by hydrogenases in green algae, was first observed over 60 years ago in Scenedesmus obliquus [1,2]. Since then, hydrogenase enzymes that either uptake or evolve H 2 have been found in many other green algae [3,4], including Chlamydomonas reinhardtii.This particular alga is capable of evolving H 2 gas in the dark [5,6] or in the light, using H 2 O [7] or starch [8,9] as the source of reductant. The reaction is catalyzed by a monomeric, 49 kDa reversible [Fe]-hydrogenase enzyme, which has been isolated to purity by Happe and Naber [10]. Other [Fe]-hydrogenases, identified in a small group of nonphotosynthetic anaerobic microbes (bacteria and pro- tists) also catalyze either H 2 production or H 2 uptake in vivo [11,12]. They play an important role in the anaerobic energy metabolism of these organisms, mainly by reoxidizing accumulated reducing equivalents. All [Fe]-hydrogenases whose X-ray structures have been analyzed to date incor- porate a [2Fe-2S] center bridged by a cysteine residue to a [4Fe-4S] center at the catalytic site (the H-cluster). It is also known that most [Fe]-hydrogenases contain additional iron-sulfur centers that act as electron relays between carrier molecules and the H-cluster [13,14]. However, the addi- tional centers are absent in the green algal enzymes [15,16]. In addition, [Fe]-hydrogenases usually exhibit high specific activity but are easily inactivated by either O 2 or CO. Green algal [Fe]-hydrogenases have been cloned and sequenced from S. obliquus [15], C. reinhardtii [16,17] and Chlorella fusca [18]. Besides the physiologically and bio- chemically characterized HydA1 [Fe]-hydrogenase [15], S. obliquus possesses a gene sequence that encodes a second polypeptide with all the essential attributes of an [Fe]- hydrogenase protein [19]. The expression of the second putative S. obliquus hydrogenase gene was shown to be constitutive (in contrast to the inducible expression of its HydA1), suggesting different physiological roles for the two enzymes. However, the expression and activities of the two hydrogenases have not been studied concomitantly in the same organism, under the same physiological conditions. Hydrogenase activity in C. reinhardtii is induced by anaerobiosis. Anaerobic states can be achieved either Correspondence to M. L. Ghirardi, National Renewable Energy Laboratory, 1617 Cole Blvd., Golden CO 80401, USA. Fax: + 1 303 384 6150; Tel.: + 1 303 384 6312; E-mail: maria_ghirardi@nrel.gov Abbreviations: CpI, one of the cloned [Fe]-hydrogenases from Clostridium pasteurianum; PSII, photosystem II; BS, basal salts; EST, expressed sequence tag; ORF, open-reading frame; PAR, photosynthetically active radiation; TAP, tris-acetate-phosphate. Accession numbers: AY055756 (C. reinhardtii HydA2 cDNA sequence); AY090770 (C. reinhardtii HydA2 promoter region and genomic DNA sequence). Note: Operated for the US Department of Energy by the Midwest Research Institute, Batelle and Bechtel under contract number DE-AC36–99G010337. *Present address: Limnological Station, Plant Biology Department, University of Zurich, Seestrasse 187, 8802 Kilchberg, Switzerland. (Received 1 April 2003, revised 29 April 2003, accepted 30 April 2003) Eur. J. Biochem. 270, 2750–2758 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03656.x physically, by purging algal cultures with a neutral gas, or physiologically, by incubating algal cultures in sulfur-free medium [20,21]. The latter condition prevents the turnover of the D1 protein of photosystem II (PSII), causes the partial inhibition of PSII, and leads to a reduction of photosynthetic O 2 evolution. The partial inhibition of PSII is sufficient to create anaerobic culture conditions and induce H 2 -production activity, which lasts for 3–4 days under continuous illumination. In this paper, we characterize a second hydrogenase gene, HydA2,inC. reinhardtii, and use the terminology estab- lished by the international hydrogenase community [12] to name both prokaryotic and eukaryotic hydrogenase genes. The primary amino-acid sequence of HydA2 (a) contains all the strictly preserved motifs present in the core, catalytic-site sequences found in algal [Fe]-hydrogenases; (b) shows high similarity to the [Fe]-hydrogenases from Clostridium pasteu- rianum [22], Trichomonas vaginalis [23], and Desulfovibrio vulgaris [24]; and (c) is 68% identical to C. reinhardtii HydA1 [16]. Additional evidence shows that HydA1 and HydA2 are encoded by different genes, HydA2 is expressed both at the transcript and protein levels, and its expression is regulated by anaerobiosis and growth conditions. We also demonstrate that both HydA1 and HydA2 transcripts are expressed under anaerobic conditions induced by sulfur deprivation. However, the expression of the two transcripts differs during the sulfur-deprivation period. This suggests differences in the regulation of expression of the two genes, also suggested by differences in their promoter regions. Our current work provides information on the expression of the two C. reinhardtii hydrogenases under three different physiological conditions. Materials and methods Cell growth and anaerobic induction Chlamydomonas reinhardtii strain 400 (cell wall-less) was grown photoheterotrophically in tris/acetate/phosphate (TAP) medium [25] supplemented with 5% (v/v) CO 2 in air or photoautotrophically on basal salts (BS) [26]. The cultures were illuminated with cool white fluorescent light (150 lEÆm )2 Æs )1 , PAR). Cells were harvested at D 750  1, corresponding to a concentration of about 15 lg chloro- phyll per mL, and the harvested cells were anaerobically induced as described previously [27] for 4–5 h. For sulfur- deprivation experiments, cells grown as above were washed with sulfur-free TAP medium and resuspended in the same medium at a final chlorophyll concentration of 20 lgÆmL )1 [20]. The cultures were then incubated in sealed glass bottles under continuous fluorescent light for up to 4 days. cDNA library screening Two specific primers, BE5P1 (5¢-AACATCTTCAAGGA GCGTGGCATC-3¢)andBE3P1(5¢-AGACAGCAGGA GACTCACAATCAC-3¢), were used to amplify a C. rein- hardtii expressed sequence tag (EST), BE337478, from a strain 21gr cDNA library kindly provided by J. Davies, Exelixis Inc., South San Francisco, CA, USA [17]. Clone EST_26,wasthenusedtogenerateanapproximately 1200 bp NotI/EcoRI restriction fragment that was dig- oxigenin-labeled and used as a probe for cDNA library screening. The HydA2 clone was retrieved in pBluescript SK(–), purified and submitted to the Iowa State University Sequencing and DNA Synthesis Facility for sequencing. Sequences were evaluated based on the chromatograms, and they were assembled using the WISCONSIN PACKAGE V.10 software by Genetics Computer Group, Inc (San Diego, CA, USA). Both strands of DNA were sequenced independently, and the sequence data for HydA2 cDNA has been deposited in GenBank under Accession No. AY055756. Isolation and sequencing of genomic HydA2 The HydA2 gene was isolated from an E. coli BAC library of C. reinhardtii chromosomal DNA (Invitrogen, San Diego, CA, USA). Probing of the filter was performed as described by the manufacturer using a HydA2-specific probe that encompassed the 5¢-UTR and approximately 150 bp of the coding region of HydA2. The probe was radiolabeled using the Rediprime random primer labeling kit (Amersham, Piscataway, NJ, USA). Four positive clones were identified and obtained as pure strains from P. Lefebvre (University of Minnesota, USA). The HydA2 portion of each BAC clone was amplified by PCR in 50 lL reactions that contained 1 lLofKOD HOTSTART polymerase (Novagen, Madison, WI, USA), 25 lL of BAC clone cell-free lysate as template, 125 ng each of forward (5¢-CTGGACGTGACAAACAAGA CCC-3¢, located at the start of the 5¢-UTR) and reverse (5¢-TGACACTGTCTGTGCG-3¢, near the stop codon) primers complementary to HydA2,1m M MgSO 4 ,0.2m M each dNTPs, and 2% (v/v) dimethylsulfoxide in 1 · reaction buffer (Novagen, Madison, WI, USA). All four clones produced a similar sized PCR product of  3.3 kb. The PCR product from clone 27d1 was gel purified and sequenced. Sequencing of the purified product was performed at Davis Sequencing, LLC. (Davis, CA, USA) on an Applied Biosystems 3730 automated sequencer. The HydA2 gene sequence has been deposited in GenBank under Accession no. AY090770. Northern blot analysis To obtain transcript hybridization signals that truly reflect the aerobic state of the algae at t ¼ 0, it proved essential to lyse noninduced algal cells as quickly as possible to avoid the establishment of anaerobiosis in the dark by respiratory O 2 consumption. Total RNA was isolated at different time points from anaerobically induced samples using the SNAP RNA Isolation Kit (Invitrogen, San Diego, CA, USA). DNA was removed by treatment with RNase-free DNaseI (0.013 units per lL). Ten micrograms of RNA were separated by electrophoresis on denaturing 1.1% (w/v) agarose, 0.22 M formaldehyde gels and then blotted onto a Nytran N + nylon membrane with 10· NaCl/Cit [28] as the transfer buffer. Radiolabeled probes specific for either HydA1 or HydA2 were generated using Rediprime random primer labeling kits (Amersham, Piscataway, NJ, USA). Denatured probes were hybridized to the membranes in prehybridization buffer [6· NaCl/Cit buffer, 5 · Den- hardt’s solution, 0.1% (w/v) SDS] overnight at 65 °C. Ó FEBS 2003 Expression of a second [Fe]-hydrogenase in C. reinhardtii (Eur. J. Biochem. 270) 2751 Following hybridization, the membranes were washed and exposed to X-ray film at )80 °C for 1–4 days. Southern blot analysis of C. reinhardtii genomic DNA for HydA1 and HydA2 Total genomic DNA was prepared from C. reinhardtii using the Qiagen DNeasy Genomic Kit (Qiagen, Valencia, CA, USA) and digested with PstI. Digested DNA (0.5 lg) was separated by agarose gel electrophoresis. Southern hybridizations were performed under identical conditions as described above for Northern hybridizations using the radiolabeled probes specific for either HydA1 or HydA2. Heterologous overexpression of HydA1 and HydA2 The HydA1 and HydA2 ORF was amplified by PCR using primer pairs containing flanking NdeI/BamHI sites (HydA1 forward 5¢-GCCGCACCCGCTGCGGAG-3¢, reverse 5¢-TCACTTCTTCTCGTCCTT-3¢; HydA2 for- ward 5¢-GCGACCGCAACTGATGCT-3¢,reverse 5¢-CTAAGCATCGGCCTCGGC-3¢). After restriction digestion, the HydA1 and HydA2 genes were cloned into the corresponding site of the pET-16b expression vector (Novagen, Madison, WI, USA) producing pET-HydA1 and pET-HydA2. The inserts of pET-HydA1 and pET- HydA2 were sequenced, confirming that the fragments contained the exact full coding region of each hydro- genase without the corresponding transit peptide sequences. The E. coli strain BL21(DE3)pLysS was transformed with both constructs pET-HydA1 and pET-HydA2. Expression was induced with 1 m M isopropyl thio-b- D -galactoside (IPTG) at an A 600 ¼ 0.3. Antibody generation and immunoblot analysis A region of low amino acid sequence homology between HydA1 and HydA2 (the insert between motifs 2 and 3) was screened for high antigenicity using two methods, Alpha Diagnostic (San Antonio, Texas) and the JaMBW online program (http://members.aol.com/_ht_a/lucatoldo/myhome page/JaMBW). Both methods identified a 14-residue long oligopeptide (VAE RLAHKVEEAAA) in HydA2 as a possible candidate. The oligopeptide was synthesized by Sigma Genosys (The Woodlands, TX, USA), coupled to the keyhole limpet hemacyanin (KLH) protein carrier and injected into rabbits to induce antibody generation. The resulting serum was immunoaffinity purified and tested for reaction against HydA1 and HydA2 overexpressed in E. coli and against HydA2 in anaerobically induced algal extracts. Algal cells were harvested during the mid-logarithmic phase by centrifugation at 2000 g for2min.Pelletswere resuspended in 50 m M Tris/HCl pH 8.5 with 20 m M sodium dithionite at  200 lg chlorophyll per mL and induced anaerobically. After induction, all steps were performed under strictly anaerobic condition. The hydrogenase-con- taining fraction was partially purified as described previ- ously [29] and run on SDS/PAGE under denaturing conditions with a 10% acrylamide gel in Tris/glycine buffer. The separated proteins were then blotted onto a poly(viny- lidene difluoride) membrane and probed with the HydA2- specific antibody. The cross-reaction was detected with a chromogenic reaction using anti-IgG secondary Igs conju- gated with alkaline phosphatase (Bio-Rad, Hercules, CA, USA). H 2 -Production assays The rates of H 2 -production were measured with a modified Clark electrode, as described previously [27]. Hydrogen production by sulfur-deprived cultures was measured by gas chromatography, using a Hewlett Packard 5890A Series II instrument equipped with a thermal conductivity detector [16]. Homology structure modeling Homology structure models of the putative C. reinhardtii HydA2 hydrogenase relative to the known structure of the C. pasteurianum CpI enzyme [30] were generated using the program SWISS - MODEL [31]. CLUSTAL W alignments of the predicted processed HydA2 sequence to CpI were used to manually optimize backbone threading. Final versions of the models were submitted to SWISS - MODEL for validation. The resulting homology structures were further refined by energy minimization with GROMOS .Calculatedrmsdvalues between the resulting HydA2 model and CpI include all shared backbone atoms. Results and discussion Genetic analysis of HydA2 The first [Fe]-hydrogenase from C. reinhardtii, HydA1,was recently cloned (GenBank, accession numbers CRE012098, AY055755, AF289201), and was shown to encode a functional enzyme [16]. The deduced amino acid sequence of the HydA1 catalytic site was further utilized in a BLAST search and revealed a close match to an expressed sequence tag (EST) from C. reinhardtii, BE33478. The EST was amplified from a cDNA library (Materials and methods). The resulting clone was identical in nucleotide sequence to BE33478. However, it was distinct from the original HydA1 amplification probe, as it contained a unique NotIrestric- tion site. This partial clone was then used as a probe to screen a cDNA library, which led to the retrieval of a full- length clone. The sequence of the retrieved clone shows an ORF for a polypeptide of 505 amino acid residues. According to the classification of hydrogenase genes reported in the review from Vignais et al.[12],theORF was termed HydA2. The HydA2 cDNA has a 139 nucleotide 5¢-UTR and an 873 nucleotide 3¢-UTR (excluding the polyadenylated tail). A polyadenylation signal (TGTAA) characteristic of nuc- lear-encoded genes in C. reinhardtii [32] is located 854 bp downstream from the stop codon, 19 bp upstream from the end of the 3¢-UTR. Figure 1 shows an amino acid sequence alignment of the translated C. reinhardtii HydA1 and HydA2 ORFs compared to S. obliquus HydA1 [15], a partial amino acid sequence for the second, highly homologous protein (which we call HydA2) found in S. obliquus [19], and Clostridium pasteurianum CpI. 2752 M. Forestier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 All the distinctive structural features of algal [Fe]- hydrogenases are also present in the C. reinhardtii HydA2 amino-acid sequence [33–35], including the well-conserved C-terminal part (C-domain) that binds the catalytic H-cluster. As seen in other algal [Fe]-hydrogenases, the N-terminal part (F-domain) of HydA2 also lacks the additional [4Fe-4S] or [2Fe-2S] centers (F-cluster) found in nonalgal [Fe] hydrogenases. The three complete motifs found in the catalytic H-cluster of [Fe]-hydrogenases, motif 1 (PMFTSCCPxW), motif 2 (MPCxxKxxExxR) and motif 3 (FxExMACxGxCV), have also been found in the algal sequences and are marked in Fig. 1. Each contains cysteine residues (*) that ligate the catalytic [4Fe-4S] center. The cysteine residue in motif 3 (#) bridges the [4Fe-4S] to the [2Fe-2S] center of the active H-cluster. Comparisons of C. reinhardtii HydA2 with the [Fe]-hydrogenases from C. reinhardtii and S. obliquus HydA1 show 68% and 61% identity, respectively. Algal [Fe]-hydrogenases also share the following charac- teristic features (Fig. 1): (a) an amino acid residue insertion (eight residues in both S. obliquus and C. reinhardtii HydA1 and HydA2) upstream of the H-cluster motif 1 and (b) a second amino acid insertion (16 residues in C. fusca, 16 residues in S. obliquus HydA1 and HydA2, 45 residues in C. reinhardtii HydA1, and 54 residues in C. reinhardtii HydA2) between H-cluster motifs 2 and 3. The biological implications of these unique features are not known at present, but, given their ubiquity in all cloned algal hydrogenases, they might be critical to specific functional or structural peculiarities of the algal enzymes. In order to determine that HydA1 and HydA2 are encoded by distinct nuclear genes, C. reinhardtii genomic DNA was purified, digested with PstI and probed sepa- rately with HydA1-andHydA2-specific probes (Southern not shown). A single PstI site is present within the HydA1 genomic sequence where the probe hybridizes. The same PstI restriction site, however, is absent from the HydA2 sequence (data not shown). As expected, the HydA1 probe detected two PstI fragments at 2.8 kb and 1.0 kb, while the HydA2 probe detected only a single fragment at 6.5 kb, clearly demonstrating that HydA1 and HydA2 are encoded by distinct DNA sequences. It was also observed that the two hydrogenase genes map on different linkage groups (L. Mets, University of Chicago, personal communication). The HydA1 gene maps on linkage group III, and HydA2 maps on linkage group IX. Finally, the two hydrogenase gene sequences are found to be present in different contigs (10 and 12, respectively) of the recently released C. rein- hardtii genome. The latter also confirmed the target- specificity of the HydA1 and HydA2 DNA probes. Algal hydrogenases are nuclear-encoded and thus must be transported into the chloroplast to function in H 2 metabolism. In eukaryotic organisms, transit signal peptide sequences direct nuclear-encoded proteins to specific organ- elles, and these sequences are then cleaved from the mature peptide. Chloroplast transit peptides are usually rich in serine, threonine, alanine and valine, but are deficient in acidic residues [36]. Moreover, chloroplast transit peptides have three characteristic domains [37] that are present in the first 63 amino acid residues of the HydA2 protein. The exact Fig. 1. Sequence alignment of the [Fe]-hydrogenases. The protein alignment was performed using the PILEUP / GENEDOC program (http://search launcher.bcm.tmc.edu/multi-align/multi-align.html). Amino acid residues highlighted in black represent identities between at least four of the hydrogenases, and those highlighted in grey show similarity between at least four of the sequences. Cr, C. reinhardtii HydA1 [16] and HydA2 (this work); So, S. obliquus HydA1 [15] and HydA2 [19]; and Cp, C. pasteurianum HydI [22]. Ó FEBS 2003 Expression of a second [Fe]-hydrogenase in C. reinhardtii (Eur. J. Biochem. 270) 2753 cleavage site recognized by chloroplast stromal processing peptidases is not known [38], but the motif VXA has been identified near the transit peptide cleavage site in a number of Chlamydomonas chloroplast-targeted proteins [39]. In HydA1, the transit peptide is cleaved one amino acid downstream from the sequence VACAA at position 56 of the nascent peptide [16]. By analogy, a VXA motif cleavage site is located at amino acid 61 of the HydA2 ORF (VAA), suggesting a cleavage site after residue 63. This cleavage site was also identified using the CHLOROP program [40]. Additional studies will be required to determine whether HydA2 is indeed processed proteolytically, and if so, where cleavage actually occurs in vivo. The isolation and sequencing of the complete HydA2 gene was done using a HydA2-specific probe (Materials and methods) to screen a BAC library of cloned C. reinhardtii genomic DNA. Following identification of four positive clones, the HydA2 region of a single BAC clone was amplified by PCR and directly sequenced (GenBank Accession number AY090770). The length of the complete HydA2 gene (from the 5¢-UTR to the 3¢-UTR minus the poly A tail) is 4.62 kb and consists of eight exons and nine introns (average intron size, 150 bp). The structural arrangement of HydA2 is thus more complex than HydA1, which contains only seven introns [16]. Interestingly, the promoter regions of HydA1 and HydA2 are unique and lack significant regions of sequence homology. Whereas the HydA2 promoter region has a characteristic TATA box located 24 bp upstream from the 5¢-UTR, the HydA1 promoter region [16] has no TATA-like sequence until 187 bp upstream from the 5¢-UTR. These differences suggest potential differences in the regulation of expression of the two genes. Gene expression during anaerobic induction The coexpression of the HydA1 and HydA2 transcripts was studied by Northern blot analysis. In Fig. 2, we used specific probes for the HydA1 (d)orHydA2 (s) transcripts, whose transcription was induced by a shift to anaerobic conditions. The transcript levels of both genes increased rapidly during the anaerobic treatment. Whereas low but detectable levels of the HydA2 transcript were observed in the BS-grown cells at t ¼ 0 (Fig. 2B, s), none were detected at the same time point in TAP-grown cells (Fig. 2A, s). In cells grown on TAP medium (Fig. 2A), the accumulation of HydA1 and HydA2 transcripts reached a maximum after about 90 min of treatment, the same time that H 2 -photoproduction activity levels reached steady-state (Fig. 2A, bars). Activity measurements were performed on the same samples at the same time that cells were harvested for mRNA extraction. IncellsgrownonBSmedium(Fig.2B),transcriptaccu- mulation and induction of H 2 -photoproduction activity occurred more slowly, reaching steady-state after Fig. 2. Effects of anaerobiosis on hydrogenase transcription, enzyme function, and transcript stability. (A) and (B) HydA1 (d)andHydA2 (s) transcript accumulation following incubation under dark, anaerobic conditions. Transcript levels were measured in C. reinhardtii cells grown on either TAP (A) or BS (B) medium and normalized to the amount of 23S rRNA of the respective sample. Simultaneous measurements of H 2 photoproduction activity were performed with the same cultures. (C) Northern blots of cultures grown on either TAP or BS medium after incubation under anaerobic conditions at 4 °C overnight (O/N), followed by exposure to O 2 for 15 min (+O 2 ).TherespectiveratesofH 2 photoproduction (lmoles H 2 per mg chlorphyll per h) are shown above each blot. 2754 M. Forestier et al.(Eur. J. Biochem. 270) Ó FEBS 2003 240–300 min of anaerobic treatment. There were no observed differences in the rate of accumulation of the two transcripts under these conditions. Induction for longer periods of time did not further change the levels of the two transcripts (not shown). The induced rates of H 2 photopro- duction vary in different experiments and reach steady-state at slightly different time points, possibly due to the different levels of anaerobic induction achieved with different cultures. However, the onset of H 2 -photoproduction acti- vity in BS-grown cells was consistently later than for TAP- grown cells. The activity data and the Northern blot analyses shown in Fig. 2 include samples from three representative experiments. Figure 2C shows that the levels of HydA1 and HydA2 transcripts in TAP-grown cultures remained high during overnight (O/N) anaerobic incubation at 4 °C and that the cultures maintained approximately 75% of their maximum H 2 -production activity. However, the same 4 °CO/N incubation of BS-grown cultures resulted in a significant decrease in the HydA2 transcript level, but no major effects on the HydA1 transcript levels or on H 2 -production activity. Exposure of the induced cultures to O 2 is known to cause alossofH 2 -photoproduction activity and a reduction in hydrogenase levels [29]. Samples of the O/N anaerobically induced cultures were tested for H 2 -photoproduction activity and transcript levels following a 15 min exposure to O 2 . Figure 2C, lane +O 2 shows that H 2 -photoproduc- tion activity in both cultures was completely lost. However, the transcripts in the photoautotrophic (BS) culture were virtually undetectable after the cells were exposed to O 2 , whereas the transcripts in the photoheterotrophic (TAP) culture were still present. Together, these data demonstrate the lower stability of the HydA2 transcript on exposure to O 2 under photoautotrophic conditions (Fig. 2C) and indi- cate that the levels of the two transcripts may be modulated by other factors in addition to O 2 . Gene expression during sulfur deprivation The expression of HydA1 and HydA2 transcripts was also studied under sulfur-deprivation conditions. In the absence of sulfur, the rates of photosynthetic O 2 evolution drop below those of O 2 consumption by respiration after about 24 h of incubation. As a consequence, sealed cultures of green algae become anaerobic in the light [41,42]. The HydA1 transcript was detected after 24 h (Fig. 3, bottom) of incubation in sulfur-depleted medium, about the time that the medium becomes anaerobic and H 2 production starts [20]. After that, the levels of HydA1 increased up to about 48 h, corresponding to the time when H 2 -production rates are high (Fig. 3, top). In contrast to HydA1, the levels of the HydA2 transcript increased up to 24 h and then gradually decrease over a total period of two days. The rates of HydA1 and HydA2 transcript accumulation under sulfur- deprivation-induced anaerobiosis clearly differ from each other (Fig. 3). Furthermore, the HydA2 transcript appears to be less stable under these conditions than HydA1 (Fig. 3), as observed in Fig. 2C. This may be the result of differences in transcriptional regulation (see above) and may signal different physiological roles for the two hydrogenases in algal metabolism. This hypothesis will be investigated in the future. The HydA2 protein is expressed during anaerobic induction In order to determine whether HydA2 is an expressed protein, HydA2-specific oligo-peptide antibody was syn- thesizedandusedforWesternblottingofC. reinhardtii extracts. To confirm the specificity of the HydA2 antibody, it was first tested on recombinant, partially purified C. reinhardtii hydrogenases. SDS/PAGE of E. coli extracts that overexpressed either HydA1 or HydA2 exhibited a Fig. 3. Induction of hydrogenase activity and gene transcription under sulfur-deprived conditions. (Top) Hydrogen-production activity of C. reinhardtii cultures incubated in sulfur-deprived TAP medium for 1–3 days in a sealed photobioreactor. The hydrogenase activity at t ¼ 0 was 0. (Bottom) RNA was isolated from cells subjected to sulfur- deprivation conditions as above, and hybridized as described in the Material and methods. The data, showing photographs of typical blots, are from one representative experiment. Fig. 4. Heterologous and homologous expression of the HydA2 protein. Western blots of IPTG-induced E. coli total protein extracts over- expressing, HydA1 or HydA2 (lanes 1 and 2, respectively) and partially purified C. reinhardtii cell extracts, either noninduced (lane 3) or anaerobically induced (lane 4). All blots were probed with the HydA2- specific antibody. The presence of a band in lane 1 above 49 kDa represents a nonspecific response seen only in the overexposed blot. It is not HydA1, as the band is also present in E. coli that had not been induced by IPTG (data not shown). Ó FEBS 2003 Expression of a second [Fe]-hydrogenase in C. reinhardtii (Eur. J. Biochem. 270) 2755 major band that migrated at the predicted mass of 49 kDa (data not shown). Whereas recombinant HydA2 was detec- ted by HydA2 antibody on the Western blot (Fig. 4, lane 2), the same antibody did not detect recombinant HydA1 (Fig. 4, lane 1). Thus, the antibody detects the HydA2 protein specifically. As expected, extracts of aerobically grown C. reinhardtii cells showed no HydA2 reactive protein (Fig. 4, lane 3). However, the antibody did recognize a protein in extracts of anaerobically induced cells, which comigrated with the recombinant HydA2 protein (Fig. 4, lane 4). These results confirm that HydA2 is expressed in C. reinhardtii and accumulates after anaerobic induction. There is precedence for multiple [Fe]-hydrogenases in different organisms, and the presence of multiple hydro- genases (both [Fe] and [NiFe]) involved in different metabolic pathways in the same organism is not unusual. For example E. coli has at least four different hydrogenases [12,43], Trichomonas vaginalis has two [23,44], and Desulf- ovibrio vulgaris has three [12]. These different hydrogenases are expressed under different conditions and catalyze either H 2 uptake or H 2 evolution. However, despite work suggesting the expression of two different hydrogenases in the green alga S. obliquus [15,19], until now no systematic studies have been done on the expression and physiological role of multiple hydrogenases in algae. The current work has addressed some of the expression issues; however, at this point, we still cannot assign a specific function for the two C. reinhardtii hydrogenases. Additional research will be carried out to specifically knock out each of the hydrogenase genes independently, so that that specific function of each gene can be investigated. Structural models of the HydA2 [Fe]-hydrogenase The alignment of the C. reinhardtii HydA2 and CpI peptide sequences (Fig. 1) reveals a high degree of primary sequence conservation (43% identity and 54% similarity). Peptide motifs that represent the active site [13,30] and the putative H 2 channel [24] located within the hydrogenase core exhibit a much higher degree of conservation (75% identity, 90% similarity). In comparison, the mature forms of the HydA1 and HydA2 peptide sequences share 81% identity and 74% similarity with each other, and in addition, 91% identity is evident for the active site and H 2 -channel motifs. In order to visualize the significance of the primary sequence homology, a theoretical model of HydA2 was generated from the solved X-ray structure of CpI. As shown in Fig. 5, HydA2 (as does HydA1, data not shown) exhibits a high degree of structural similarity to CpI (rmsd of 1.55 A ˚ , 1480 backbone atoms). The predicted locations of HydA2 (and HydA1, data not shown) peptide sequence motifs that represent the active site and H 2 -channel match closely to the positions of the corresponding residues in CpI. The rmsd between the HydA2 and CpI core regions is 0.74 A ˚ over 1336 shared atoms (334 Ca atoms). This information corroborates our assignment of a hydrogenase function to HydA2, provides us with a model to compare the catalytic functions of isolated HydA1 and HydA2 in the future, and serves as a guide to future site-directed mutagenesis studies of the two algal hydrogenase proteins. In conclusion, we have cloned and sequenced a second [Fe]-hydrogenase gene from the green alga, C. reinhardtii. The promoter regions of the two algal hydrogenase genes exhibit significant differences that may reflect differences in the regulation and/or roles of the two hydrogenases in algal physiology, as has been observed in other multiple hydro- genase systems. The transcription of HydA1 and HydA2 in response to the removal of O 2 depends on the composition of the growth medium (photoheterotrophic vs. photoauto- trophic; sulfur-replete vs. sulfur-deprived). These observa- tions underpin the importance of hydrogenases for algal metabolism, and will spur further research on the specific physiological and biochemical pathways related to each hydrogenase in C. reinhardtii. Acknowledgements We thank Dr John Davis, Exelixis Inc. for the cDNA library, Prof Lauren Mets, University of Chicago, for providing us with his mapping results, and Scott Plummer, a graduate student from the Colorado School of Mines for his help in screening the cDNA library Fig. 5. Homology structure models of the C. reinhardtii HydA2 hydrogenase. A model of the predicted structure of HydA2 is shown compared to the X-ray structure of CpI [30]. The H-clusters are identified in CPK colors as space-filled atoms. The backbone colors correspond to secondary structure type (red, a-helix; cyan, b-sheet; grey, random coil). The nonconserved N-terminal domain of CpI is also colored grey, and the Fe-S centers are represented as ball and stick diagrams. 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Biochem. 270) Ó FEBS 2003 . Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions Marc Forestier 1, *, Paul King 1 , Liping Zhang 1 ,. potential differences in the regulation of expression of the two genes. Gene expression during anaerobic induction The coexpression of the HydA1 and HydA2