Characterization of a mutant of Chlamydomonas reinhardtii deficient in the molybdenum cofactor.
Molybdenum (Mo) is an essential micronutrient for almost all organisms. In eukaryotes, it forms a part of the molybdenum cofactor (Moco), which is required for numerous enzymes involved in carbon, nitrogen and sulfur metabolism. Mo is taken up by cells in the form of molybdate and recently molybdate transporters have been identified in Arabidopsis thaliana and Chlamydomonas reinhardtii. Here, we report the characterization of a novel mutant (DB6) of C. reinhardtii generated by random insertional mutagenesis that is unable to assimilate nitrate as a nitrogen source because it lacks functional nitrate reductase (NR). Besides lacking NR, DB6 also lacks xanthine dehydrogenase activity; a common requirement of both enzymes is Moco. DB6 displays a 'molybdate-repairable' phenotype--growth on nitrate is partially restored by supplementing media with high levels of molybdate. This phenotype is typically associated with mutants defective in either molybdate transport or insertion of Mo into the pterin precursor of Moco. Mo content was found to be significantly lower in DB6 than in the wild-type strain, AOXR1, which suggests that DB6 is defective in Mo uptake. Genetic complementation with a variety of candidate genes that include the known molybdate transporter MOT1 and DNA that spans the site of mutation was unable to recover the wild-type phenotype. Taken together, our results indicate that DB6 is a novel molybdate transport-deficient mutant.
Li
Wenze
W
Department of Biology and The Biotron, University of Western Ontario, London, ON, Canada N6A 5B7.
Fingrut
Daniel R
DR
Maxwell
Denis P
DP
D020418:Algal Proteins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics*; D003067:Coenzymes / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D016254:Mutagenesis, Insertional; D009154:Mutation; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010641:Phenotype; D011621:Pteridines / Q000378:metabolism; D048030:RNA, Algal / Q000235:genetics
D020418:Algal Proteins; D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D050901:Nitrate Reductase; D009584:Nitrogen; D011621:Pteridines; D048030:RNA, Algal; C044659:molybdate; C022261:molybdopterin