ISOLATION AND CHARACTERIZATION OF 2 NEW NEGATIVE REGULATORY MUTANTS FOR NITRATE ASSIMILATION IN CHLAMYDOMONAS-REINHARDTII OBTAINED BY INSERTIONAL MUTAGENESIS

Plasmid DNA carrying either the nitrate reductase (NR) gene or the arg ininosuccinate lyase gene as selectable markers and the corresponding Chlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based o n their chlorate sensitivity in the presence of ammonium. Among 8975 t ransformants, two mutants, N1 and T1, were obtained. Genetic character ization of these mutants indicated that they carry recessive mutations at two different loci, named Nrg1 and NrgZ. The mutation in N1 was sh own to be linked to the plasmid insertion. Two copies of the nitrate r eductase plasmid, one of them truncated, were inserted in the N1 genom e in inverse orientation. In addition to the chlorate sensitivity phen otype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (C-14-methylammonium) transport, as we ll as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest that Nrg1 and Nrg2 are regulatory genes whi ch specifically mediate the negative control exerted by ammonium on th e nitrate assimilation pathway in C. reinhardtii.