Tài liệu Báo cáo khoa học: Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions doc

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:
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 ( />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. Domains unique to HydA2 and not found in CpI are rep-
resented in green. Images of the structures were made with
VIEWERLITE

software (Accelrys).
2756 M. Forestier et al.(Eur. J. Biochem. 270) Ó FEBS 2003
for the HydA2 clone. MF would like to acknowledge a grant to
prospective researchers from the Swiss National Science Foundation.
MS and MLG were supported by the U.S. DOE Hydrogen program.
TH thanks the Japanese New Energy and Industrial Technology
Development Organization (NEDO-project no. 01GB1) for financial
support.
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