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.
- Chlamydomonas reinhardtii
- H2 photoproduction
- insertional mutagenesis
- metalloprotein assembly
Hydrogen has enormous potential to serve as a non-polluting 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 CO-oxidation, 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 . 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 hydrogen-photoproduction mutants
To identify genes necessary for hydrogenase activity in C. reinhardtii, we screened an insertional mutagenesis library (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 . 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 down-regulates 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 . Among the former, is the starchless mutant sta7-10 from the National Renewable Energy Laboratory .
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 compared with WT control cells. Measurements of light-induced, 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 sta7-10 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  that encodes a unique radical S-adenosylmethionine protein [16,17]. This protein is required for the assembly of an active [FeFe]-hydrogenase . 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. Full-length 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 , 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.
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 co-expression 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.).
International Hydrogenases Conference 2004: Independent Meeting held at the University of Reading, 24–29 August 2004. Edited by R. Cammack (King's College London, U.K.) and F. Sargent (University of East Anglia, Norwich, U.K.). Organized by R. Cammack and R. Robson (University of Reading, U.K.). Sponsored by COST (European Cooperation in the field of Scientific and Technical Research), the European Science Foundation and the European Office of Aerospace Research and Development.
Abbreviations: WT, wild-type
- © 2005 The Biochemical Society