Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha.
Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
Pignocchi
Cristina
C
Laboratorio di Genetica Microbica, Dipartimento di Biotecnologie Agrarie ed Ambientali, Universit degli Studi di AnconaVia Brecce Bianche, 1-60131 AnconaItaly.
Berardi
Enrico
E
Laboratorio di Genetica Microbica, Dipartimento di Biotecnologie Agrarie ed Ambientali, Universit degli Studi di AnconaVia Brecce Bianche, 1-60131 AnconaItaly.
Cox
Brian S
BS
Laboratorio di Genetica Microbica, Dipartimento di Biotecnologie Agrarie ed Ambientali, Universit degli Studi di AnconaVia Brecce Bianche, 1-60131 AnconaItaly.
D002704:Chlorates / Q000494:pharmacology; D003470:Culture Media; D004352:Drug Resistance, Microbial; D004579:Electron Transport; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005800:Genes, Fungal; D009154:Mutation / Q000235:genetics*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics*; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification
D002704:Chlorates; D003470:Culture Media; D009566:Nitrates; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D009584:Nitrogen