The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3 (-), the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.