Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster

Abstract

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 FM was estimated. In uptake assays, the crnAI mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnAI reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction. One of the most thoroughly studied metabolic processes in eucaryotic microorganisms is ni

Nitrate transporters and peptide transporters

“…The NRT2 family of high affinity nitrate transporters was first discovered in the chlorate-resistant mutant, crnA, now renamed NRTA, of Aspergillus nidulans: the nitrate uptake defect of this mutant is seen in the conidiospore and young mycelia stages, but not in older mycelia [52,53]. Subsequent searches led to the identification of an equivalent gene family in Chlamydomonas [54], marine cyanobacterium [55], and a variety of plants, including barley [56], tobacco [57], soybean [58], and Arabidopsis [30,38].…”

Test of Interaction Between Genetic Markers That Affect Fitness in Aspergillus niger

“…Since most active uptake systems are organized similarly, interactions between the five auxotrophic mutations may be relevant for the fitness of Aspergillus under growth limiting conditions. The ern marker of A. niger that we used is probably similar to the crnA marker in A. nidulans (Brownlee and Arst 1983), which causes a nitrate permease with a lower affinity for chlorate, and in young mycelium also for nitrate. Therefore, ern can be considered as a "leaky" auxotrophic mutation.…”

C‐terminal truncation of the transcriptional activator encoded by area in Aspergillus nidulans results in both loss‐of‐function and gain‐of‐function phenotypes

“…Crude extracts were prepared by homogenization of mycelia in a micro-homogenizer for 6min in the appropriate extraction buffer as described by Shaffer and Arst (1984) and centrifugation at 27000 X g for 30 min, the supernatant being taken for enzyme assay and soluble protein determination. The extraction buffer for nitrate reductase was that of Brownlee and Arst (1983) whilst that for nitrite reductase contained (final concentrations) 50mM sodium pyrophosphate, 33% (v/v) glycerol, 1 mM FAD, 1 mM PMSF, 1 mM OL-dithiothreitol. lOmM ethytene glycol bis (p-aminoethyl ether) ft/,W,W,W-tetraacetic acid and lOmM EDTA(disodium salt) at pH 8.6.…”

“…lOmM ethytene glycol bis (p-aminoethyl ether) ft/,W,W,W-tetraacetic acid and lOmM EDTA(disodium salt) at pH 8.6. Assays of NADPH; nitrate oxidoreductase (EC 1.6.6.3) and NADPH: nitrite oxidoreductase (EC 1.6.6.4) were performed as described by Brownlee and Arst (1983) except that (final concentration) lOmM sodium sulphite was included in nitrate reductase assay mixtures to inhibit nitrite reductase (according to Garrett and Cove, 1976). Because ot the lability of nitrite reductase, it was assayed within 15 min of obtaining the cell-free extract.…”