Abstract
The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.
Highlights
Nitrate is a major nitrogen source for most micro-organisms and plants
Higher plants including crop plants have numerous copies, e.g. Arabidopsis thaliana has seven members of the NRT2 gene subfamily and multiple copies of the NRT1 subfamily (Okamoto et al, 2003), the latter group belonging to the related major facilitator superfamily (MFS) family of protondependent oligopeptide transporter family (POT:TC 2.A.17)
Results indicated nitrate transport by NrtA and NrtB exhibited different sensitivities to chlorate and caesium, while a range of other chemicals with comparable structures to nitrate had no significant effect on transport
Summary
The transport of nitrate (and nitrite) by most bacteria, algae, fungi and plants across membrane barriers is carried out mainly by high-affinity transporters (Trueman et al, 1996). Such high-affinity transporters belong to the nitrate/nitrite porter family, NNP: TC 2.A.1.8 (www.tcdb.org; Pao et al, 1998; Forde, 2000), a subfamily forming a distinct cluster of the largest secondary transporter family, the major. Higher plants including crop plants have numerous copies, e.g. Arabidopsis thaliana has seven members of the NRT2 gene subfamily and multiple copies of the NRT1 subfamily (Okamoto et al, 2003), the latter group belonging to the related MFS family of protondependent oligopeptide transporter family (POT:TC 2.A.17). Nitrate transport in a WT strain of A. nidulans has been shown to be protondependent (Downey & Gedeon, 1994), and transport of nitrate by NrtA has been shown to be energized by protons (Boyd et al, 2003)
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