In Chlamydomonas reinhardtii, hydrogenase enzymes in the photosynthetic pathway produce molecular hydrogen (H₂) under anaerobic conditions.  Expression of transcripts from the two genes encoding hydrogenase (HYDA1 and HYDA2) is repressed in aerobic conditions(1, 2).  To study the molecular mechanism of hydrogenase gene repression, we have created reporter gene constructs to assay the activity of the promoter regions of HYDA1 and HYDA2.   The coding sequence of the PF14 gene, required for flagellar motility and swimming, was fused downstream of HYDA1 and HYDA2 promoter sequences.  When transformed into mutant (immotile) pf14 cells, the pHYDA1::PF14 and pHYDA2::PF14 constructs produced transformants with a conditional swimming phenotype inducible by an anaerobic environment.  The presence of the reporter gene in the transformants was verified by PCR as well as by Southern blot analysis.  The results suggest that the HYDA gene expression is regulated at the level of transcription and the promoter sequences contain elements required for induction of hydrogenase gene expression in anoxic conditions.   To further characterize and quantify the activity of these promoter elements, we have fused the promoters to a second reporter gene encoding luciferase from Renilla reniformis (Rluc).  In vitro mutagenesis of the promoter region of the chimeric gene constructs will allow us to identify the DNA structural elements required for regulation of transcription of the HYDA genes by oxygen.
Ref: 1. Happe and Kaminski (2002) Eur. J. Biochem. 269, 1022-1032
       2. Forestier et al. (2003) Eur. J. Biochem. 270, 2750-2758