The bZIP Transcription Factor MeaB Mediates Nitrogen Metabolite Repression at Specific Loci

ABSTRACT In Fusarium fujikuroi, bikaverin (BIK) biosynthesis is subject to repression by nitrogen. Unlike most genes subject to nitrogen metabolite repression, it has been shown that transcription of bik biosynthetic genes is not AreA dependent. Searching for additional transcription factors that may be involved in nitrogen regulation, we cloned and characterized the orthologue of Aspergillus nidulans meaB, which encodes a bZIP transcription factor. Two transcripts are derived from F. fujikuroi meaB: the large transcript (meaB L) predominates under nitrogen-sufficient conditions and the smaller transcript (meaB S) under nitrogen limitation, in an AreA-dependent manner. MeaB is specifically translocated to the nucleus under nitrogen-sufficient conditions in both F. fujikuroi and A. nidulans. Deletion of meaB resulted in partial upregulation of several nitrogen-regulated genes, but only in the ΔmeaB ΔareA double mutant were the bikaverin genes significantly upregulated in the presence of glutamine. These data demonstrate that MeaB and AreA coordinately mediate nitrogen metabolite repression and, importantly, that independently of AreA, MeaB can mediate nitrogen metabolite repression at specific loci in F. fujikuroi.

Negative Roles of a Novel Nitrogen Metabolite Repression-Related Gene, TAR1, in Laccase Production and Nitrate Utilization by the Basidiomycete Cryptococcus neoformans

ABSTRACT The multicopper oxidase laccase is widespread in fungi and has great industrial importance. One puzzle regarding laccase production in the basidiomycetous yeast Cryptococcus neoformans is that it is inhibited by high temperature (e.g., 37°C). In this paper, we report identification of a nitrogen metabolite repression-related gene, TAR1, which is responsible for laccase repression. Disruption of TAR1 results in a significant increase in the level of LAC1 mRNA at 37°C. The putative protein Tar1 shares a moderate level of similarity with the nitrogen metabolite repressors Nmr1 and NmrA from Neurospora crassa and Aspergillus nidulans, respectively. Likewise, Tar1 has a negative role in the utilization of nitrate. Furthermore, the structure of Tar1 is unique. Tar1 lacks the long C-terminal region of Nmr1 and NmrA. It contains the canonical Rossmann fold motif, GlyXXGlyXXGly, whereas Nmr1 and NmrA have variable residues at the Gly positions. Interestingly, the promoter region of TAR1 contains three TTC/GAA repeats which are likely the heat shock factor (Hsf) binding sites, implying that Hsf has a role in laccase inhibition. TAR1 mediation of temperature-associated repression of LAC1 suggests a novel mechanism of laccase regulation and a new function for Nmr proteins. Our work may be helpful for industry in terms of promotion of laccase activity.

AgtA, the Dicarboxylic Amino Acid Transporter of Aspergillus nidulans, Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis

ABSTRACT We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

A Gene from Aspergillus nidulans with Similarity to URE2 of Saccharomyces cerevisiae Encodes a Glutathione S-Transferase Which Contributes to Heavy Metal and Xenobiotic Resistance

ABSTRACT Aspergillus nidulans is a saprophytic ascomycete that utilizes a wide variety of nitrogen sources. We identified a sequence from A. nidulans similar to the glutathione S-transferase-like nitrogen regulatory domain of Saccharomyces cerevisiae Ure2. Cloning and sequencing of the gene, designated gstA, revealed it to be more similar to URE2 than the S. cerevisiae glutathione S-transferases. However, creation and analysis of a gstA deletion mutant revealed that the gene does not participate in nitrogen metabolite repression. Instead, it encodes a functional theta class glutathione S-transferase that is involved in resistance to a variety of xenobiotics and metals and confers susceptibility to the systemic fungicide carboxin. Northern analysis showed that gstA transcription is strongly activated upon exposure to 1-chloro-2,4-dinitrobenzene and weakly activated by oxidative stress or growth on galactose as a carbon source. These results suggest that nitrogen metabolite repression in A. nidulans does not involve a homolog of the S. cerevisiae URE2 gene and that the global nitrogen regulatory system differs significantly in these two fungi.