Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 417, Issue 2, Pages 704-709Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2011.12.002
Keywords
Algae; Chlamydomonas; [FeFe]-hydrogenase; HYDA; Photosynthesis
Categories
Funding
- US AFOSR [FA9550-05-1-0365, FA9550-11-1-0211]
- NASA [NNGO5GL52H]
- NSF [0824469, 0951094]
- US DOE Office of Science [DE-AC3699G010337]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [951094, 0824469] Funding Source: National Science Foundation
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Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H-2 chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H-2 production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H-2 photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H-2 production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H-2-photoproduction rates did not exceed those of wild-type cells. (C) 2011 Elsevier Inc. All rights reserved.
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