Generation and characterization of high CO2 requiring mutants in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii (referred to as C. reinhardtii hereinafter) possesses a CO2 concentrating mechanism (CCM) that allows the alga to grow at low CO2 concentrations (100 ppm CO2 in air). This dissertation represents the results from the characterization of three CCM mutants, as well as generation of eleven more potential mutants deficient in the CCM. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron dense structure within the chloroplast. The first mutant characterized has a highly disorganized pyrenoid. The results indicated that the gene disrupted in this mutant, CIA6, is required for the formation of the pyrenoid. Furthermore this showed that the loss of the pyrenoid correlated with the loss of the CCM. These results supported the hypothesis that the pyrenoid is required for a functional CCM. A second mutant investigated was a revertant of a mutant in the gene PGP1. Initially, the pgp1 strain could not grow under low CO2. But over time, it regained the ability to grow under low CO2 conditions. Data is presented that shows the change in growth phenotype is a result of a second site reversion. The results from this study suggested that another phosphoglycolate phosphatase (PGP2) might play a role in the phenotype reversion. Thirdly, a cell wall deficient strain CC-503 was found to be missing a periplasmic carbonic anhydrase (CAH1). The possible reason for the loss of CAH1, and the resulting consequences of losing CAH1 were investigated in this strain. Lastly, a PCR-based reverse genetics mutagenesis screen was performed to identify more genes involved in the CCM in C. reinhardtii. Eleven potential mutants were isolated, and it was shown that this method could be used on a larger scale in the future to generate mutants missing key CCM genes.