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CrnA encodes a nitrate transporter in Aspergillus nidulans.

ABSTRACT: The nucleotide sequence of the Aspergillus nidulans crnA gene for the transport of the anion nitrate has been determined. The crnA gene specifies a predicted polypeptide of 483 amino acids (molecular weight 51,769). A hydropathy plot suggests that this polypeptide has 10 membrane-spanning helices with an extensive hydrophilic region between helices six and seven. No striking homology was observed between the crnA protein and other reported membrane proteins of either prokaryotic or eukaryotic organisms, indicating that the crnA transporter may represent another class of membrane protein. Northern blotting results with wild-type cells show that (i) control of crnA expression is subject to nitrate (and nitrite) induction as well as nitrogen metabolite repression and (ii) regulation of the crnA gene is exerted at the level of mRNA accumulation, most likely at transcription, in response to the nitrogen source in the growth medium. Furthermore, similar studies with mutants of nirA and areA control genes and the niaD nitrate reductase structural gene show that crnA expression is mediated by the products of nirA (nitrate induction control gene), areA (nitrogen metabolite repression control gene), and niaD (involved in autoregulation of nitrate reductase).

SUBMITTER: Unkles SE

PROVIDER: S-EPMC50778 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

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crnA encodes a nitrate transporter in Aspergillus nidulans.

Unkles S E SE Hawker K L KL Grieve C C Campbell E I EI Montague P P Kinghorn J R JR

Proceedings of the National Academy of Sciences of the United States of America 19910101 1

The nucleotide sequence of the Aspergillus nidulans crnA gene for the transport of the anion nitrate has been determined. The crnA gene specifies a predicted polypeptide of 483 amino acids (molecular weight 51,769). A hydropathy plot suggests that this polypeptide has 10 membrane-spanning helices with an extensive hydrophilic region between helices six and seven. No striking homology was observed between the crnA protein and other reported membrane proteins of either prokaryotic or eukaryotic or ...[more]

PMID: 1986367

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Project description:1. In Aspergillus nidulans nitrate and nitrite induce nitrate reductase, nitrite reductase and hydroxylamine reductase, and ammonium represses the three enzymes. 2. Nitrate reductase can donate electrons to a wide variety of acceptors in addition to nitrate. These artificial acceptors include benzyl viologen, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, cytochrome c and potassium ferricyanide. Similarly nitrite reductase and hydroxylamine reductase (which are possibly a single enzyme in A. nidulans) can donate electrons to these same artificial acceptors in addition to the substrates nitrite and hydroxylamine. 3. Nitrate reductase can accept electrons from reduced benzyl viologen in place of the natural donor NADPH. The NADPH-nitrate-reductase activity is about twice that of reduced benzyl viologen-nitrate reductase under comparable conditions. 4. Mutants at six gene loci are known that cannot utilize nitrate and lack nitrate-reductase activity. Most mutants in these loci are constitutive for nitrite reductase, hydroxylamine reductase and all the nitrate-induced NADPH-diaphorase activities. It is argued that mutants that lack nitrate-reductase activity are constitutive for the enzymes of the nitrate-reduction pathway because the functional nitrate-reductase molecule is a component of the regulatory system of the pathway. 5. Mutants are known at two gene loci, niiA and niiB, that cannot utilize nitrite and lack nitrite-reductase and hydroxylamine-reductase activities. 6. Mutants at the niiA locus possess inducible nitrate reductase and lack nitrite-reductase and hydroxylamine-reductase activities. It is suggested that a single enzyme protein is responsible for the reduction of nitrite to ammonium in A. nidulans and that the niiA locus is the structural gene for this enzyme. 7. Mutants at the niiB locus lack nitrate-reductase, nitrite-reductase and hydroxylamine-reductase activities. It is argued that the niiB gene is a regulator gene whose product is necessary for the induction of the nitrate-utilization pathway. The niiB mutants either lack or produce an incorrect product and consequently cannot be induced. 8. Mutants at the niiribo locus cannot utilize nitrate or nitrite unless provided with a flavine supplement. When grown in the absence of a flavine supplement the activities of some of the nitrate-induced enzymes are subnormal. 9. The growth and enzyme characteristics of a total of 123 mutants involving nine different genes indicate that nitrate is reduced to ammonium. Only two possible structural genes for enzymes concerned with nitrate utilization are known. This suggests that only two enzymes, one for the reduction of nitrate to nitrite, the other for the reduction of nitrite to ammonium, are involved in this pathway.

Project description:BACKGROUND: The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. RESULTS: In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. CONCLUSIONS: This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed mutagenesis. Investigation into the area of sugar transport in fungi presents a crucial step for improving the S. cerevisiae xylose metabolism. Moreover, we have demonstrated that the introduction of adaptive mutations beyond the introduced xylose utilization genes is able to improve S. cerevisiae xylose metabolism.

Dataset Information

CrnA encodes a nitrate transporter in Aspergillus nidulans.

Publications

crnA encodes a nitrate transporter in Aspergillus nidulans.

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