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, 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 H
2
-production does not occur under photosynthetic
O
2
-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)
Eur. J. Biochem. 269, 1022–1032 (2002) ÓFEBS 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 biological H
2
evolution 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].
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) medium
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 the
algae were anaerobically adapted by flushing the solution
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 anaero-
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 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 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)-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-select
TM
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 °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 PCR conditions were altered to increase the
specificity of the amplification. The PCR 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 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.
ÓFEBS 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 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
)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 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
5·10
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 method [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 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 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¢-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 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 pellet was resuspended in solubilization 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 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 sequences 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 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
Akgt11 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
1024 T. Happe and A. Kaminski (Eur. J. Biochem. 269)ÓFEBS 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 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 in
E. 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 +
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.
ÓFEBS 2002 Fe-hydrogenase from Chlamydomonas reinhardtii (Eur. J. Biochem. 269) 1025
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
codonaredrawninboxes.Boldfaceletters
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.
1026 T. Happe and A. Kaminski (Eur. J. Biochem. 269)ÓFEBS 2002