Nitrate Reductase Deficient Mutants of Chlamydomonas reinhardii. Biochemical Characteristics

Abstract

Two mutants of Chlamydomonas reinhardii that cannot grow with nitrate as nitrogen source have been examined biochemically. Each mutant differs from the wild-type strain by a single mutation in a Mendelian gene. One mutant (14/15) carries a mutation in a gene designated nitB. This organism took up nitrate and converted nitrate-nitrogen to insoluble-nitrogen; nevertheless no enzymic activities associated with nitrate reductase (NADH-nitrate reductase, reduced benzyl viologen nitrate reductase or inducible NADH-cytochrome c reductase) could be demonstrated in frozen/thawed cells or cell-free extracts. At high nitrate concentrations (5 mM) the rate of nitrate uptake by this mutant [V max 59 nmol (mg dry wt)–1 h–1] was considerably lower than that of wild-type [V max 920 nmol (mg dry wt)–1 h–1]. It is concluded that nitB is probably a regulatory gene for nitrate reductase. The other mutant (17/4) has a mutation in a gene designated nitA. This organism did not take up nitrate and had no NADH-nitrate reductase or inducible NADH-cytochrome c reductase activities. Frozen/thawed cells and cell-free extracts had reduced benzyl viologen nitrate reductase activity. The enzyme from the nitA mutant eluted from a Sepharose 4B column later than wild-type enzyme; it was therefore probably of lower molecular weight. The nitA mutation of C. reinhardii closely resembles the nit-3 mutation of Neurospora crassa and, like this mutation, it probably results in loss of the NADH combining sub-unit of nitrate reductase. Strain 137c of C. reinhardii had no enzymic activities associated with nitrate reductase and did not take up nitrate. This organism may have mutations in both the nitA and nitB loci.