Impact of ammonium permeases mepA, mepB, and mepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi

Abstract

In Fusarium fujikuroi, the production of gibberellins and bikaverin is repressed by nitrogen sources such as glutamine or ammonium. Sensing and uptake of ammonium by specific permeases play key roles in nitrogen metabolism. Here, we describe the cloning of three ammonium permease genes, mepA, mepB, and mepC, and their participation in ammonium uptake and signal transduction in F. fujikuroi. The expression of all three genes is strictly regulated by the nitrogen regulator AreA. Severe growth defects of DeltamepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In DeltamepB mutants, nitrogen-regulated genes such as the gibberellin and bikaverin biosynthetic genes are derepressed in spite of high extracellular ammonium concentrations. mepA mepB and mepC mepB double mutants show a similar phenotype as DeltamepB mutants. All three F. fujikuroi mep genes fully complemented the Saccharomyces cerevisiae mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, whereas only MepA and MepC restored pseudohyphal growth in the mep2/mep2 mutant. Overexpression of mepC in the DeltamepB mutants partially suppressed the growth defect but did not prevent derepression of AreA-regulated genes. These studies provide evidence that MepB functions as a regulatory element in a nitrogen sensing system in F. fujikuroi yet does not provide the sensor activity of Mep2 in yeast, indicating differences in the mechanisms by which nitrogen is sensed in S. cerevisiae and F. fujikuroi.

FIG. 1.

Dendrogram of aligned protein sequences of known mammalian, plant, bacterial, and fungal permeases of the MEP/AMT family or putative ammonium permeases chosen by homology. The following are accession numbers for the indicated proteins: Homo sapiens RH type B, AAG01086; Mus musculus rh50, AAC25155; Escherichia coli AMTA, AAA97110; Brassica napus AMT1, AAG28780; Arabidopsis thaliana AMT1, P54144; Lotus japonicus AMT1, AAG24944; Triticum aestivum AMT1, AAS19466; Dictyostelium discoideum AMTB, BAB39710; Caenorhabditis elegans AMT1, P54145; C. elegans AMT2, Q20605; Agrobacterium tumefaciens AMTB, AAL43739, E. coli AMTB, AAD14837; D. discoideum AMTA, BAB39709; S. cerevisiae Mep1, P40260; S. cerevisiae Mep3, P53390; H. cylindrosporum AMT3, AAK82417; Aspergillus nidulans MeaA, EAL73117; Aspergillus fumigatus MeaA, EAL87679; Schizosaccharomyces pombe hypothetical protein SPAC664.14, CAB65815; Cryptococcus neoformans Mep1, AAW40795; U. maydis Ump1, AAL08424; H. cylindrosporum AMT1, AAM21926; H. cylindrosporum AMT2, AAK82416; C. neoformans putative ammonium transporter, AAW45844; U. maydis Ump2, AAO42611; Microbotryum violaceum MepA, AAD40955; A. fumigatus ammonium transporter, EAL90420; A. nidulans MepA, AAL73118; F. fujikuroi MepB; T. borchii AMT1, AAL11032; N. crassa MepA, CAD21326; F. fujikuroi MepA; S. cerevisiae Mep2, P41948; Candida glabrata unnamed protein, XP_447968; C. albicans hypothetical protein CaO19.13117, XP_713400; Pichia angusta AMM1p, AAQ76838; A. fumigatus ammonium transporter EAL91508; Phytophthora infestans ammonium transporter, AAN31513.

FIG. 2.

Transmembrane structure of MepA, MepB, and MepC. Data were obtained from TMHMM2 (20). All three proteins consist of 11 transmembrane helices and 10 loops of denoted length (ll).

FIG. 3.

Expression analysis of the F. fujikuroi ammonium permease genes mepA, mepB, and mepC in the wild-type IMI58289 and the ΔglnA and ΔareA mutant strains. The fungal strains were cultivated for 5 days in ICI medium with 10 mM glutamine, and after a 2-h incubation in nitrogen-free ICI medium, the washed mycelia were shifted into medium without nitrogen, with 10 mM ammonium nitrate, or with 10 mM glutamine.

FIG. 4.

Complementation of the yeast mep1 mep2 mep3 triple mutant with the F. fujikuroi mepA, mepB, and mepC cDNA fragments under the control of the S. cerevisiae GAL1 promoter. All three mep genes fully restored the growth of the mep1 mep2 mep3/mep1 mep2 mep3 mutant on SLADG minimal medium. Transformation of the diploid yeast mep2/mep2 mutant with the F. fujikuroi mepA and mepC genes, but not with the F. fujikuroi mepB gene, restored pseudohyphal growth on the nitrogen limited medium SLADG.

FIG. 5.

The Mep proteins of F. fujikuroi mediate [¹⁴C]methylamine uptake. Rate of uptake versus substrate concentration curves for MepA (A), MepB (B), and MepC (C) when proteins are expressed from the GAL1-10 promoter in a S. cerevisiae mep1 mep2 mep3 triple mutant strain (see Material and Methods).

FIG. 6.

Strategy for generating the replacement vectors pΔmepA, pΔmepB, and pΔmepC which were used for deletion of the corresponding ammonium permease genes. mepB and mepA were replaced by the nourseothricin and mepA by the hygromycin resistance cassettes. Introns are shown as gray bars. One of the flanks of each replacement vector was used as a probe for hybridization in Southern blotting. For confirmation of homologous integration of the replacement cassette into the corresponding mep locus, the genomic DNA of the wild type and the deletion mutants was digested with SphI (mepA and mepB) or with EcoRI (mepC). In all three cases, the loss of the wild-type (WT) fragment is shown.

FIG. 7.

Plate assays for monitoring the growth of the wild-type and mutant strains on ICI medium with different ammonium citrate concentrations. (A) Growth of single and double ammonium permease deletion strains on different ammonium citrate concentrations (0, 1, 10, and 100 mM). The circles around the wild type (WT), mepA, and mepC mutants show the borders of the colonies on plates without any nitrogen. mepB single and ΔmepA ΔmepB (ΔmepA/B) and ΔmepB ΔmepC (ΔmepB/C) double mutants are not able to grow on this medium. (B) Biomass production of the wild type (WT) and the five mutant strains in ICI medium with 10 mM ammonium sulfate or 10 mM glutamine after 72 h of incubation.

FIG. 8.

Production of bikaverin and GA3 by the wild-type strain IMI58289; the ΔmepA, ΔmepB, and ΔmepC single mutants; and the ΔmepA ΔmepB (mepA/B) and ΔmepB ΔmepC (mepB/C) double mutants. The strains were cultivated in a time course for 3 days in medium containing 12 mM ammonium sulfate or 12 mM glutamine. The pigmentation of the culture fluids demonstrates the much earlier start of the bikaverin production in the ΔmepB single and double mutants and also in the ΔmepC mutant with ammonium as the nitrogen source. The GA3 production under the same conditions is shown by TLC. As for the bikaverin production, the GAs were produced much earlier (at 30 h) in the ΔmepB single and double mutants, whereas the production starts only at 90 h in the wild type and ΔmepA mutant.

FIG. 9.

Northern blot analysis with the F. fujikuroi wild type (WT); the ΔmepA, ΔmepB, and ΔmepC single mutants; and the ΔmepA ΔmepB (ΔmepA/B) and ΔmepB ΔmepC (ΔmepB/C) double mutants. Strains were cultivated in a time course for 3 days in medium containing 12 mM ammonium sulfate or glutamine. For the glutamine cultures, only one time point (72 h) is shown due to the low expression levels of the GA and bikaverin genes at 24 and 48 h. The filters were probed with the cDNA fragments of the genes.

FIG. 10.

Northern blot analysis with the F. fujikuroi wild type (WT) and ΔgdhA mutant. Strains were cultivated in a time course for 3 days in medium containing 10 mM ammonium sulfate. The mycelia were washed and shifted for 2 h into nitrogen-free medium or medium containing 10 or 100 mM ammonium sulfate, with or without the GS inhibitor MSX (2 mM). The filters were probed with the cDNA fragments of the GA (cps/ks and P450-4) and bikaverin (MO and pks4) biosynthetic genes as well as the three mep genes and the GS-encoding gene glnA.

FIG. 11.

Overexpression of mepC (glnA_prom::mepC) in the ΔmepB deletion mutant. (A) Growth of several transformants carrying the overexpression cassette on 1 mM ammonium sulfate. Transformants T1, T2, T3, and T10 were chosen for detailed analyses (see below). Transformant T5 does not contain the whole promoter-gene fusion fragment and shows the same growth defect as the ΔmepB deletion mutant (negative control). (B) Bikaverin production in the wild type, ΔmepB mutant, and four mepC-overexpressing transformants, which still show a much earlier pigment production than the wild type. (C) Northern blot analysis with the F. fujikuroi wild type, the ΔmepB mutant, and four mepC-overexpressing transformants. The strains were cultivated for 24 h in medium containing 12 mM ammonium sulfate. The early pigmentation (as shown in panel B) corresponds very well with early expression of several AreA target genes (for an explanation, see the legend of Fig. 10) in the mepC-overexpressing transformants. WT, wild type.

FIG. 12.

Model of the nitrogen regulation network in F. fujikuroi. MepB is postulated as a key component in this model, acting as the major ammonium permease. In addition, mepB might also act as a nitrogen sensor either by mediating the signal of nitrogen availability or indirectly by causing a drop in the intracellular glutamine level. In the latter case we suggest an intracellular sensor transducing the glutamine signal to AreA via TOR and/or additional signaling pathways. WT, wild type.