Download Identification of genes required for hydrogenase activity in

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Biochemical Society Transactions (2005) Volume 33, part 1
Identification of genes required for hydrogenase
activity in Chlamydomonas reinhardtii
M.C. Posewitz*†, P.W. King*, S.L. Smolinski*, R. Davis Smith II*, A.R. Ginley*1 , M.L. Ghirardi* and M. Seibert*2
*National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401, U.S.A., and †Department of Environmental Science and Engineering,
Colorado School of Mines, Golden, CO 80401, U.S.A.
Abstract
The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction
catalysed by an [FeFe]-hydrogenase. To identify genes that influence H2 production in C. reinhardtii, a
library of 6000 colonies on agar plates was screened with sensitive chemochromic H2 -sensor films for clones
defective in H2 production. Two mutants of particular interest were fully characterized. One mutant, hydEF-1,
is unable to assemble an active [FeFe]-hydrogenase. This is the first reported C. reinhardtii mutant that is not
capable of producing any H2 . The second mutant, sta7-10, is not able to accumulate insoluble starch and has
significantly lowered H2 -photoproduction rates in comparison with the wild-type. In hydEF-1, anaerobiosis
induces transcription of the two reported C. reinhardtii hydrogenase genes, HydA1 and HydA2, indicating a
normal transcriptional response to anaerobiosis. In contrast, the transcription of both hydrogenase genes in
sta7-10 is significantly attenuated.
Introduction
Hydrogen has enormous potential to serve as a nonpolluting fuel, alleviating the environmental and political
concerns associated with fossil energy utilization. Among
the most efficient H2 -generating catalysts are the hydrogenase
enzymes [1,2] that are found in nature in many taxonomically
diverse microorganisms [2–4]. Several microbes have been
investigated for possible production of H2 [4–7]. These
organisms have the ability to produce H2 anaerobically in
pathways coupled either to dark fermentation, dark COoxidation, light-dependent N2 -fixation or photosynthetic
water oxidation.
Photosynthetic microalgae are unique among the diversity
of organisms capable of H2 production. In these microbes,
H2 production can be coupled directly to water oxidation
through photosystem II and the photosynthetic electron
transport chain, providing the means to generate H2 by
directly using sunlight [4,6]. Algal H2 production was first
reported by Gaffron and Rubin, in seminal experiments
performed over 60 years ago [8]. Over the past several
years, great strides have been made in sustaining H2 photoproduction activity in the green alga, Chlamydomonas
reinhardtii [6,9], and intensive research continues to probe
novel ways to use this and other photosynthetic organisms
to generate renewable H2 .
Identification of hydrogenphotoproduction mutants
To identify genes necessary for hydrogenase activity in
C. reinhardtii, we screened an insertional mutagenesis library
Key words: Chlamydomonas reinhardtii, H2 photoproduction, hydrogenase, insertional mutagenesis, metalloprotein assembly, starch.
Abbreviation used: WT, wild-type.
1
2
Present address: University of California, Berkeley, CA 94720, U.S.A.
To whom correspondence should be addressed (email Mike [email protected]).
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Biochemical Society
(kindly provided by Professor A. Melis, University of
California at Berkeley) for mutants with attenuated levels
of H2 photoproduction using sensitive chemochromic H2 sensor films [10,11]. Screening DNA insertional mutagenesis
libraries in C. reinhardtii has become a popular strategy
for identifying important genes involved in specific cellular
pathways and processes [12]. Mutants were generated by
transforming the Arg7 gene into C. reinhardtii strain CC425,
which is an arginine auxotroph. The Arg7 gene is randomly
incorporated into the C. reinhardtii genome and disrupts
small sections of WT (wild-type) genomic DNA. An
example of a mutant isolated by this procedure is shown in
Figure 1.
A total of 22 positive clones have been identified so far from
over 6000 insertional mutants. The phenotype of all positive
clones was further assayed in liquid algal suspensions. Fourteen of the selected clones exhibited H2 -photoproduction
rates that are at least 50% less than the background strain
after 5 h of dark, anaerobic induction. Eight of these
strains have rates less than 10% of the WT under the assay
conditions used. The identified mutants can be classified
generally as strains with a metabolic defect that downregulates the transcription of the two reported C. reinhardtii
hydrogenase genes, HydA1 and HydA2 [13,14], and mutants
that are unable to accumulate an active hydrogenase enzyme
[10]. Among the former, is the starchless mutant sta7-10 from
the National Renewable Energy Laboratory [11].
Hydrogenase activity is attenuated
in the starchless mutant
The sta7-10 mutant contains a disrupted isoamylase gene
that is required for the accumulation of insoluble starch
in C. reinhardtii [11,15]. The sta7-10 mutant contains less
than 3% of the glucose found in insoluble starch when
International Hydrogenases Conference 2004
Figure 1 A chemochromic sensor (upper panel) after illumination
of candidate C. reinhardtii colonies on a TAP agar plate
(lower panel)
The dark blue spots on the sensor (upper panel) are indicative of H2 detection. The third colony from the left could not produce sufficient H2 to
elicit a colorimetric response, which indicates a H2 -production mutant.
compared with WT control cells. Measurements of lightinduced, H2 -production rates showed that the sta7-10 mutant
is able to photoproduce H2 at low rates after a short period
of anaerobic induction, followed by a gradual decrease in
the rates up to 7 h of incubation. The parental strain, on the
other hand, shows maximum H2 -photoproduction activity
after 3–4 h of anaerobic induction. The maximal WT rates
are approx. 5-fold greater than the maximal H2 -production
rates observed in the sta7-10 mutant. Northern-blot analysis
indicates that the mRNA transcripts for both the HydA1 and
HydA2 [FeFe]-hydrogenase genes are expressed in the sta710 mutant at levels greater than WT, 0.5 h after anaerobic
induction. However, after 1.5 h, transcript levels of both
HydA1 and HydA2 begin to decrease rapidly and reach nearly
undetectable levels after 7 h. In WT cells, the hydrogenase
transcripts accumulate more slowly, reach a plateau after
4 h of anaerobic treatment and maintain the same level of
expression for more than 7 h under anaerobic incubation.
Complementation of mutant cells with genomic DNA
corresponding to the STA7 gene reverses both the starch
accumulation and H2 -production phenotypes. The results
indicate that STA7 and starch metabolism play an important
role in C. reinhardtii H2 photoproduction. Moreover, the
results indicate that mere anaerobiosis is not sufficient to
maintain hydrogenase-gene expression without the underlying physiology, an important aspect of which is starch
metabolism. This important result suggests that, besides
anaerobiosis, the transcription of the algal hydrogenase genes
is regulated by other unknown metabolic factors.
HydEF and HydG are required to assemble
an active [FeFe]-hydrogenase
Another National Renewable Energy Laboratory mutant
(hydEF-1) lacks a functional HydEF gene [10] that encodes
a unique radical S-adenosylmethionine protein [16,17].
This protein is required for the assembly of an active
[FeFe]-hydrogenase [10]. The hydEF-1 strain is the only
characterized C. reinhardtii mutant that is unable to produce
any H2 at all.
In hydEF-1, anaerobiosis induces transcription of the
two reported C. reinhardtii hydrogenase genes, indicating
a normal transcriptional response to anaerobiosis. Fulllength hydrogenase protein is also detected by Western-blot
analysis in the hydEF-1 mutant. However, no detectable
hydrogenase activity is seen in hydEF-1 cultures that have
been induced anaerobically. The HydEF gene, as well as
another hydrogenase-related radical S-adenosylmethionine
gene that we have isolated, HydG [10], are anaerobically
induced concomitantly with the two hydrogenase structural
genes in WT cultures. In the hydEF-1 mutant, all of
these genes are induced anaerobically with the exception
of HydEF, which is disrupted. These results indicate that the
necessary transcriptional, regulatory and signalling pathways
in the hydEF-1 mutant remain intact during anaerobiosis. The
hydEF mutation specifically disrupts hydrogenase activity,
and this mutant has no discernable difference in phenotype
compared with the WT when grown aerobically under low
light.
Complementation of the C. reinhardtii hydEF-1 mutant
with genomic DNA corresponding to a functional copy of
the HydEF gene restores hydrogenase activity. Moreover, the
co-expression of the C. reinhardtii HydEF, HydG and
HydA1 genes in Escherichia coli results in the formation of an
active HydA1 enzyme in this bacterium. HydEF and HydG
represent the first reported accessory genes that are required
for the maturation of an active [FeFe]-hydrogenase in any
organism.
Conclusions
Screening of C. reinhardtii mutants was successfully used
to identify genes important for hydrogenase activity in
C. reinhardtii. Identification of the metabolic and accessory
genes required for H2 production in this organism will facilitate efforts aimed at engineering organisms with more robust
H2 -production capabilities. It is now evident that strategies
to heterologously express or manipulate the expression of
[FeFe]-hydrogenases will need to also incorporate the coexpression of the required assembly genes.
We thank Professor A. Melis for kindly providing us with the
C. reinhardtii insertional mutagenesis library. This work was
supported by the Division of Energy Biosciences, Office of Science,
U.S. Department of Energy (to M.L.G. and M.S.) and by the Hydrogen,
Fuel Cells, and Infrastructure Technologies Program, U.S. Department
of Energy (to M.L.G. and M.S.).
References
1 Frey, M. (2002) ChemBioChem 3, 153–160
2 Vignais, P.M., Billoud, B. and Meyer, J. (2001) FEMS Microbiol. Rev. 25,
455–501
3 Tamagnini, P., Axelsson, R., Lindberg, P., Oxelfelt, F., Wunschiers, R. and
Lindblad, P. (2002) Microbiol. Mol. Biol. Rev. 66, 1–20
4 Boichenko, V.A., Greenbaum, E. and Seibert, M. (2004) in
Photoconversion of Solar Energy, Molecular to Global Photosynthesis
(Archer, M.D. and Barber, J., eds.), pp. 397–452, Imperial College Press,
London
5 Hansel, A. and Lindblad, P. (1998) Appl. Microbiol. Biotechnol. 50,
153–160
C 2005
Biochemical Society
103
104
Biochemical Society Transactions (2005) Volume 33, part 1
6 Ghirardi, M.L., Zhang, L., Lee, J.W., Flynn, T., Seibert, M., Greenbaum, E.
and Melis, A. (2000) Trends Biotechnol. 18, 506–511
7 Maness, P.C., Smolinski, S., Dillon, A.C., Heben, M.J. and Weaver, P.F.
(2002) Appl. Environ. Microbiol. 68, 2633–2636
8 Gaffron, H. and Rubin, J. (1942) J. Gen. Physiol. 26, 219–240
9 Melis, A., Zhang, L., Forestier, M., Ghirardi, M.L. and Seibert, M. (2000)
Plant Physiol. 122, 127–136
10 Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and
Ghirardi, M.L. (2004) J. Biol. Chem. 279, 25711–25720
11 Posewitz, M.C., Smolinski, S.L., Kanakagiri, S., Melis, A., Seibert, M. and
Ghirardi, M.L. (2004) Plant Cell 16, 2151–2163
C 2005
Biochemical Society
12 Tam, L.W. and Lefebvre, P.A. (1995) Methods Cell Biol. 47, 519–523
13 Happe, T. and Kaminski, A. (2002) Eur. J. Biochem. 269, 1022–1032
14 Forestier, M., King, P., Zhang, L., Posewitz, M., Schwarzer, S., Happe, T.,
Ghirardi, M.L. and Seibert, M. (2003) Eur. J. Biochem. 270, 2750–2758
15 Mouille, G., Maddelein, M.L., Libessart, N., Talaga, P., Decq, A., Delrue, B.
and Ball, S. (1996) Plant Cell 8, 1353–1366
16 Sofia, H.J., Chen, G., Hetzler, B.G., Reyes-Spindola, J.F. and Miller, N.E.
(2001) Nucleic Acids Res. 29, 1097–1106
17 Nicolet, Y. and Drennan, C.L. (2004) Nucleic Acids Res. 32, 4015–4025
Received 30 September 2004