Isolation and characterization of nitrite-reductase-deficient mutants of Chlorella sorokiniana (strain 211-8k)

Abstract.

 A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO₃ ⁻ metabolism. Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO₃ ⁻, NO₂ ⁻ or NH⁺ ₄ as N-source. The mutants isolated absorb NO₃ ⁻ with rates close to those measured for the wild type and they excrete NO₂ ⁻ into the medium. The ratio between NO₃ ⁻ uptake and NO₂ ⁻ excretion was 1:1. The sensitivity of NO₃ ⁻ uptake to NH⁺ ₄ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wild-type) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds.