Báo cáo Y học: Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii pot

Differential regulation of the Fe-hydrogenase during anaerobicThomas Happe and Annette Kaminski Botanisches Institut der Universita¨t Bonn, Germany Chlamydomonas reinhardtii, a unicellul

Differential regulation of the Fe-hydrogenase during anaerobic

Thomas Happe and Annette Kaminski

Botanisches Institut der Universita¨t Bonn, Germany

Chlamydomonas reinhardtii, a unicellular green alga,

con-tains a hydrogenase enzyme, which is induced by

anaer-obic adaptation of the cells Using the suppression

subtractive hybridization (SSH) approach, the differential

expression of genes under anaerobiosis was analyzed

A PCR fragment with similarity to the genes of bacterial

Fe-hydrogenases was isolated and used to screen an

anaerobic cDNA expression library 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 transcription 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 protein The Fe-hydrogenase

contains a short transit peptide of 56 amino 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 amino acids, which are strictly conserved in the active site (H-cluster) of Fe-hydrogenases, have been identified The N-terminus of the C reinhardtii protein is markedly truncated compared

to other nonalgal Fe-hydrogenases Further conserved cysteines that coordinate additional Fe–S-cluster in 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 algae respond to anaerobic stress by switching 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 prokaryotes [1,2] and some unicellular eukaryotic

organisms [3,4] With respect to the metal 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 hydrogen-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 structure 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] cluster bridged by the sulfur atom of a cysteine residue to a unique binuclear iron subcluster [17]

Fe-hydrogenases from green algae mediate a light driven hydrogen evolution after an anaerobic adaptation [4], but this H2-production does not occur under photosynthetic

O2-evolving conditions [18,19] The electrons can be supplied by metabolic oxidation of organic compounds with the release of carbon dioxide [20,21] This light dependent electron transport is 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-insensitive and requires only pho-tosystem I activity [22] The role of the hydrogenase in green algae growing under photosynthetic conditions in the natural environment has been unclear for a long time Recently it was shown that sulfur deprivation in C rein-hardtii cultures caused anaerobic conditions and, as a consequence, hydrogen production [23,24] Under an anaerobic atmosphere, the hydrogen metabolism is the only pathway for the algae to create enough ATP, which is required for the survival under this stress 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 acetate phosphate;

DIG, digoxygenin.

Definitions: PS indicates photosystem I and II including the reaction

centers P 700 and P 680 ; Q and Z are the primary electron acceptors of the

PS II or PS I, respectively; PQ refers to the plastoquinone pool.

Note: the authors would like to dedicate this paper to Herbert Bo¨hme,

who has retired because of a malignant disease.

Note: the nucleotide sequence reported in this paper has been

submitted to the GenBank/EBI Data Bank with accession

number CRE012098.

(Received 18 June 2001, revised 17 December 2001, accepted

18 December 2001)

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 biological H2evolution in

green algae, all attempts to isolate the hydrogenase gene

from C reinhardtii have 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 The

differ-ential regulation of protein biosynthesis during anaerobic

adaptation is discussed based on Northern blot analysis

The results present fundamental data for studying the

hydrogen metabolism in photosynthetic eukaryotes On

the basis of this research, we have recently published the

isolation and characterization of the hydA gene from

the green alga Scenedesmus obliquus [27]

M A T E R I A L S A N D M E T H O D S

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(m2Æs))1

Cultures containing TAP (Tris acetate phosphate) medium

were flushed vigorously with air containing 5% CO2 Cells

were harvested by centrifugation (8 min, 5000 g) in the

mid-exponential growth stage (1–2· 106cellsÆmL)1) The pellet

was resuspended in 0.02 vol of fresh TAP medium and the

algae were anaerobically adapted by flushing the solution

with argon in the dark

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 mM), Na2S2O4

(20 mM), methyl viologen (5 mM), was incubated

anaero-bically at 25°C for 20 min One unit is defined as the

amount of hydrogenase evolving 1 lmol H2Æmin)1

Purification of the Fe-hydrogenase and amino-acid

sequence

Cells from a 40-L culture of C reinhardtii were harvested by

ultra filtration through an Amicon Ultrafiltration System

DC 10 LA, equipped with a hollow-fiber filter The pellet

was resuspended in 200 mL TAP medium After anaerobic

adaptation by flushing the solution with argon for 1 h in the

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 fragments on an SDS polyacrylamide gel, four

peptides were blotted onto a poly(vinylidene difluoride) membrane and were sequenced Automated Edman degra-dation was performed with an Applied Biosystem 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 Johannaccord-ingmeier & Howell [29] Poly(A)+RNA was isolated using the RNA Kit (Qiagen); 10 lg total RNA or 0.5 lg poly(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)-dUTP 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-selectTM cDNA Subtraction Kit (Clontech Laboratories Enc., Palo Alto, CA, USA) according to the 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 tester cDNAs were denatured separately for the first hybridization at 100°C for

30 s and then incubated for 10 h at 68°C For the second hybridization, driver cDNA was denatured at 100°C for

30 s, then directly added to the pooled mix of the previous hybridization, and incubated at 68°C for 20 h Primary and secondary PCR conditions were altered to increase the specificity of the amplification The PCR conditions with subtracted cDNA were as follows: 25 cycles each 94°C for

30 s, 68°C for 30 s, and 72 °C for 1 min The subtracted cDNA was subjected to a second round of nested PCR,

Fig 1 Schematic 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 noted.

using the same PCR conditions with a decreased number

of 15 cycles Specific primers were used for the identification

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, taking 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)1of each

primer were used; 35 cycles (denaturing at 95°C for 40 s,

annealing at 54°C for 1 min, and extension at 72 °C for

1 min) The amplified cDNA fragments were cloned into

the T overhang vector pGEMÒ-T Easy (Promega)

Screening of the cDNA library, cloning and sequencing

A cDNA library was constructed using 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 contained

5· 106recombinant phages and was amplified according to

the manufacturer’s instructions

A 366-bp PCR fragment was radiolabeled with

[a-32P]dCTP using the random-primer method [31]

Approximately 5· 105plaques 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

T7 sequencing Kit (Pharmacia Biotech) Both strands of

genomic and cDNA of hydA were completely sequenced

using a nested set of 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 of the hydA transcript

Expression studies inE 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 NdeI and BamHI

restric-tion sites, respectively (underlined) The oligonucleotide

sequences were: HydNde (5¢-CATATGGCCGCACCCG

CTGCGGAGGCGCCT-3¢), HydBam (5¢-CCGGATCC

TCAAGCCTCTGGCGCTCCTCA-3¢)

The hydA gene, corresponding to amino acids 57–497, was amplified, confirmed by sequences analysis and cloned into corresponding sites of the pET9a expression vector (Pro-mega) The constructed plasmid was then transformed into

E coli strain BL21(DE3) After induction with 1 mM

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 pellet was resuspended in solubilizaadapta-tion buffer and incubated with vigorous vortexing at RT for 30 min The protein extracts from C reinhardtii and E coli were

separat-ed by 12% SDS/PAGE and blottseparat-ed 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 sequences were analyzed with the programsSCI ED CENTRAL(Scientific Educational Software, Durham, NC, USA) andCLUSTALW[33] TheBLASTserver [34] of the National Center for Biotechnology Information (Bethseda, MD, USA) was used for database searches

R E S U L T S

Isolation of cDNA clones, which are differentially expressed during anaerobic adaptation

In order to amplify a part 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 fragments 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 correspond to any entries in the databases Four of these novel clones showed differences in expression between aerobically grown and anaerobically adapted cul-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 constructed using 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

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-32P]dCTP and used to screen 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 acids (Fig 1) The

cDNA also contained a 5¢ UTR (158-bp) and a longer 3¢

UTR (747-bp excluding 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] The promoter region does not 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 Meanwhile parts of the cDNA sequence were

determined by another group and deposited under accession

number AF289201

Southern hybridization experiments were performed at

high stringency using a PCR fragment as probe (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 is probably cleaved by a stromal peptidase 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, the localization of the mature protein in the chloroplast stroma is indicated by a high content of hydroxylated and basic amino acids in the transit 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 of S obliquus [27], which was recently isolated on the basis of the data of this work Comparisons with NiFe-hydrogenases of bacteria (including the photosynthetic cyanobacteria) had obviously lower scores, e.g 25% similarity with the NiFe-hydrogenase (HoxH) of Ralstonia eutropha [1]

A conserved domain 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 catalytic 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 acids of HydA proteins probably define a binding pocket surrounding the active 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, Lys172, 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 that Fe-hydrogenases in 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 or other conserved cysteines is completely miss-ing in HydA of C reinhardtii A novel electron transport pathway is postulated from the exogenous donor (ferred-oxin) directly to the H-cluster

Protein sequencing of the enzyme and recombinant expression of HydA inE coli

To verify that the hydA ORF encodes the 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 cDNA clones generated

from Chlamydomonas reinhardtii by suppression subtractive

hybridiza-tion (SSH) –, novel sequence +, only 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 +

4 369 Ribosomal protein L17 1.2 +

8 317 Ribosomal protein S8 0.8 +

9 184 Malate-dehydrogenase 1.8 –

10 412 Ribosomal protein S15 0.7 +

12 243 Ribosomal protein L12 0.9 –

14 272 Ribosomal protein S8 0.8 +

15 251 Ribosomal protein L37 0.7 –

19 273 Ribosomal protein S18 0.8 +

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.

hydrogen, when 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 Nucleotide 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 codon are drawn in boxes Boldface letters indicate the cDNA sequence Gray shadows mark amino acids corresponding to polypep-tide sequences that were determined by sequencing the N-terminus of the protein Black shadows mark the putative transit peptide, and the underlined amino acids indi-cate the putative cleavage site for the endo-peptidase Boldface double underlined letters indicate a signal for polyadenylation.

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 Stokkermans et al [40] and Voordouw

et al [41] who also detected no H2-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 system 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 min of anaerobiosis (Fig 6) The size

of the transcript (2.4 kb) confirmed the full-length of the isolated hydA cDNA fragment

D I S C U S S I O N

Differentially expressed genes during anaerobic adaptation

Hydrogen metabolism induced by anaerobic conditions is well established in green algae In the absence of oxygen,

C reinhardtii, and also plants, switch their metabolism to fermentation [43,44] In the light, algae degrade cellular starch via glycolysis [45] and hydrogen gas is evolved It has been suggested that reducing equivalents from the glycolysis

or the citric acid cycle can transfer their 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 advantage 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 proteins takes place under phosphate starvation Moreover, two of the identified cDNAs encode 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 an increased mRNA level Maize (Zea mays L.) responds to anaerobic stress by redirect-ing the synthetic machinery towards the synthesis of some 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) and 10 lg of DNA was loaded

per lane The DIG-dUTP labeled DNA fragment (750-bp) was used

for hybridization as indicated in Fig 1.

Fig 4 Multiple sequence alignments of Fe-hydrogenases Sequence comparison of the deduced amino-acid sequence of HydA from C reinhardtii with Fe-hydrogenases published for other organisms The protein alignment was done by using the Vector NTI program ( INFORMAX ) Black highlighted letters indicate amino acids identical to the HydA protein Gray shadowed amino acids indicate conserved changes of 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 pasteurianum [12]; N o., Nyctotherus ovalis [10] Black arrows indicate conserved cysteines, gray ones the residues that are necessary for the formation of the H-cluster and white arrows refer to the conserved cysteines of the F-cluster, which is lacking in C reinhardtii and S obliquus.

(AAPAAAEAPLSHVQQALAELAKPKD) from the

purified native enzyme [4] Further evidence that the isolated

cDNA encodes an Fe-hydrogenase is the fact that the

recombinant HydA specifically reacts with the antibodies

raised against the active enzyme The amino-acid sequence

of HydA shows only considerable similarity to

Fe-hydro-genases but not to NiFe-hydroFe-hydro-genases The Fe-hydrogenase

family is one class 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 protons 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 arranged as a [4Fe)4S] subcluster bridged to a

[2Fe] subcluster by a single cysteinyl sulfur The [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 be 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 were 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 detected 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 the electron donor (mostly ferredoxin) to the active center [56] It has been suggested that the proteins containing two [4Fe–4S] clusters are 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 missing in the algal hydrogenases The native protein of C reinhardtii is located in the chloroplast stroma [26] The first 56 amino acids of the unprocessed enzyme probably function as a transit peptide, because they were not characterized in the 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 H2-evolution, we have shown that the hydrogenase activity is directly linked to the 47.5-kDa subunit [4] As we have not found a second subunit necessary for hydrogenase activity, we suggest that

a direct electron transfer from PetF to HydA 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 spinach 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 the pET9a vector The HydA protein was overexpressed upon

induction with isopropyl thio-b- D -galactoside Lanes 1, purified

hydrogenase from C reinhardtii; Lanes 2, protein extract from 2-h

induced E coli cells was separated on an SDS polyacrylamide gel The

molecular mass marker (Bio-Rad) indicates relative molecular masses

in kDa (A) The SDS polyacrylamide gel was stained with Coomassie

Blue (B) Western blotting and immunodetection was carried out as

described previously [24].

Fig 6 Differential expression of the hydA gene shown by Northern blot analysis The aerobically grown C reinhardtii cells were centrifuged, 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 as described 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 the left (C) Immunoblot with affinity-purified antibodies on the right.

Why do the photosynthetic green algae still keep the

anaerobically induced hydrogenases? The most likely

explanation is that the enzymes ensure the survival of the

cells under these anaerobic conditions Melis et al have

shown that H2-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 reinhardtiiis still able to photoproduce hydrogen when

photosystem II is inhibited by DCMU, but no H2

-evolu-tion occurs after an addi-evolu-tion of

2,5-dibromo-3-methyl-6-isopropyl-p-benzochinon (DBMIB; Fig 7) [27] Under

anaerobic conditions, accumulated reducing equivalents

from the fermentative metabolism cannot be oxidized via

respiration, as the electron acceptor oxygen is missing The

NAD(P)H reductase protein complex has recently been

isolated from plants [58], and inhibitor experiments have

shown evidence of a membrane-bound, chloroplast-located

reductase in C reinhardtii [59] The light-dependent electron

transport of the H2-evolution is driven by plastoquinone

and photosystem I The donor ferredoxin transfers electrons

in a last step to the hydrogenase and molecular hydrogen is

released (Fig 7)

Regulation ofhydA 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 is likely that the

induction of hydA is regulated on the level of transcription

We observed that the amount of mRNA increased directly

with the measured H2-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 30 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 moment it is not clear if this effect rests upon a new synthesis or a higher stability of the hydA mRNA

As with other nuclear genes, the promoter region of the hydAfrom 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 genetic analyses are necessary to investigate the rapid induction of hydA in C reinhardtii

A C K N O W L E D G E M E N T S

The authors wish to thank D Naber for helpful advice and discussions.

We thank Dr R Deutzmann (Universita¨t Regensburg) for determina-tion of the amino-acid sequences This work was supported by the Deutsche Forschungsgemeinschaft (Ha 2555/1-1).

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