Isolation and characterisation of Chlamydomonas reinhardtii mutants with an impaired CO2-concentrating mechanism.

In order to identify new genes involved in the carbon-concentrating mechanism of Chlamydomonas reinhardtii (Dangeard), high-CO2-requiring mutants were isolated by insertional mutagenesis after transformation of strain CC1618 with a plasmid carrying Arg7 as a selectable marker gene. Six mutants were classified by their growth behaviour under ambient CO2, the affinity of the CO2-concentrating mechanism for inorganic carbon and the expression of known low-CO2-inducible proteins. The mass-spectrometric measurement of carbonic anhydrase activity and CO2/HCO3- transport revealed that four of the mutants are unable to induce a high-affinity carbon-concentrating mechanism. The expression of various carbonic anhydrases and chloroplast inner envelope polypeptides was examined with Western Blots. While three high-CO2-requiring mutants showed abnormal expression patterns, one matched the wild type. With Southern blots the total number and structure of the insertion events were determined to select possible candidates for plasmid recovery. Abnormal structures of thylakoid lamellae traversing the pyrenoid were detected by electron microscopy in some of the high-CO2-requiring mutants. Our characterisations of the insertionally generated mutants revealed phenotypes that have not been published before and therefore might be useful tools to obtain new insights on the molecular background of the CO2-concentrating mechanism and its regulation.