Using an RSP3 reporter gene system to investigate molecular regulation of hydrogenase expression in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii evolves H₂ gas under anaerobic conditions in reactions catalyzed by hydrogenase enzymes. Expression of the HYDA genes and related genes is regulated by environmental conditions including anoxia. To study the pathways of gene regulation, we utilized the reporter gene RSP3, encoding a radial spoke protein required for flagellar motility. Promoter/5'UTR sequences of HYDA1 and HYDA2 genes were fused to the RSP3 coding sequence and the constructs were transformed into immotile cells lacking a functional RSP3 gene. The resulting transformants express the RSP3 reporter under the transcriptional control of the HYDA1 or HYDA2 promoter/5'UTR sequences. They are paralyzed in the presence of O₂, but motile in anoxic conditions. To identify cis-elements or trans-acting factors that regulate gene expression in response to hypoxia, the conditionally swimming transformant strains were mutagenized and constitutively swimming strains were selected under aerobic conditions. Molecular and genetic analyses showed that mutations in both cis-elements and trans-acting factors lead to constitutive motility. Some mutant strains contain transposable elements inserted into the HYDA promoter/5'UTR sequences. These interrupted regions might indicate regulatory sequences involved in the response to hypoxia or perhaps the presence of sequences within transposable elements that stimulate transcription of the reporter gene.