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Eukaryotic Cell, February 2008, p. 187-201, Vol. 7, No. 2
1535-9778/08/$08.00+0 doi:10.1128/EC.00351-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Julian C. Rutherford,2,
Marieke Wottawa,1,
Joseph Heitman,2 and
Bettina Tudzynski1*
Institut für Botanik der Westfälischen Wilhelms-Universität Münster, Schloβgarten 3, D-48149 Münster, Germany,1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina2
Received 27 September 2007/ Accepted 14 November 2007
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ABSTRACT |
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mepB mutants on low-ammonium medium and methylamine uptake studies suggest that MepB functions as the main ammonium permease in F. fujikuroi. In mepB 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 mepB 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 mepB 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. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Under nitrogen starvation conditions, the rice pathogenic fungus F. fujikuroi produces the red pigment bikaverin and gibberellins (GAs), mainly the gibberellic acids GA3, GA4, and GA7, the causative agents of the "bakanae" disease of rice seedlings. GAs are isoprenoid plant hormones used as plant growth regulators in agriculture and horticulture (40). Bikaverin is a polyketide which is responsible for the deep red color exhibited under certain growth conditions by cultures of some Fusarium species (23). Interestingly, neither of these secondary metabolites contains nitrogen, and they do not have any obvious function in nitrogen metabolism (reviewed in reference 51). Both the GA and bikaverin biosynthetic genes are strictly repressed by nitrogen. For the GA biosynthetic genes a direct dependence on AreA has already been shown (33, 46, 47, 50), whereas the role of AreA in regulation of bikaverin genes is not yet clear.
Additional components of nitrogen metabolism also influence the correct expression of the GA and bikaverin biosynthetic genes in F. fujikuroi. Glutamine synthetase (GS) is required for the wild-type expression of a variety of genes, including those involved in ribosome biogenesis and translation initiation, which are induced in a mutant strain lacking GS (46). Surprisingly, GS activity is also required for the expression of the GA and bikaverin biosynthetic genes, which is abolished, rather than induced, in a GS null mutant (46). This suggests an important role for GS in control of cellular metabolism and highlights the complexity of the regulatory networks that respond to nitrogen metabolism in F. fujikuroi.
The GA and bikaverin biosynthetic genes are also targets of the TOR signaling pathway (47). In yeast, the conserved TOR (target of rapamycin) kinases play a significant role in nutrient sensing and cell growth by affecting nuclear localization of transcription factors, such as Gln3, and thereby controlling genes subject to NCR (reviewed in reference 41). In F. fujikuroi, several AreA-regulated genes, e.g., GA and bikaverin biosynthetic genes, are only partially derepressed by rapamycin suggesting that additional regulatory pathways involved in nitrogen metabolism exist.
We are interested in identifying components that mediate nitrogen sensing and which act upstream of the transcription factor AreA in F. fujikuroi. In yeast and filamentous fungi, nitrogen permeases, such as ammonium and amino acid permeases, can form part of the nitrogen regulatory network (15). Ammonium transport in fungi is mediated by permeases that belong to the conserved AmtB/Mep family of proteins that are related to the mammalian rhesus blood group antigens. These proteins are highly conserved within bacteria, plants, and animals and share a similar protein structure (13, 16, 18, 29, 31, 49). Within certain fungal species, one of the permeases has evolved a regulatory function. Examples include Mep2 (S. cerevisiae and Candida albicans), Amt1 (Hebeloma cylindrosporum), and Ump2 (Ustilago maydis), which are required for the induction of filamentous growth under low-nitrogen conditions (3, 18, 24, 25, 44, 52). In S. cerevisiae, this dimorphic transition requires the cooperation of two signaling pathways, the mitogen-activated protein kinase and the cyclic AMP-dependent pathways (22).
It is not clear at present the extent to which regulatory ammonium permeases are conserved within fungal species. In this study we have characterized three members of the AmtB/Mep family from F. fujikuroi that we have designated MepA, MepB, and MepC. Phenotypic and methylamine uptake studies in S. cerevisiae confirm that they are functional ammonium permeases. The genes encoding these permeases are under AreA-mediated nitrogen metabolite repression control. MepA and MepC but not MepB fully restored pseudohyphal growth in the S. cerevisiae mep2 mutant. MepB is probably the major ammonium permease in F. fujikuroi, and its deletion results in derepression of the GA and bikaverin biosynthetic genes and other genes subject to nitrogen metabolite repression under ammonium-sufficient conditions. Furthermore, overexpression of mepC in the mepB background partially suppresses the strong growth defect but not the derepression of AreA target genes on high ammonium concentrations. We suggest, therefore, a sensing or regulatory role of MepB in addition to its function as a permease.
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MATERIALS AND METHODS |
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For DNA isolation and protoplasting, F. fujikuroi strains were incubated in complete medium (39) at 28°C on a rotary shaker at 200 rpm for 3 days or 18 h, respectively. For analysis of GA production, the fungus was grown for 5 days at 28°C on a rotary shaker (190 rpm) in a liquid production medium containing 60 g/liter sunflower oil, 15 g/liter corn steep solids (Sigma, Germany), 1.0 g/liter glutamine, and 1.0 g/liter KH2PO4 or in ICI medium (14) with 20% of the ammonium nitrate concentration (20% ICI). The S. cerevisiae strains MLY40
(ura3-52 MAT), MLY131 (mep1::LEU2 mep2::LEU2 mep3::G418 ura3-52 MAT), MLY97a/ (ura3-52/ura3-52 leu2::hisG/leu2::hisG MATa/), and MLY108a/ (mep2::LEU2/mep2::LEU2 ura3-52/ura3-52 leu2::hisG/leu2::hisG MATa/) were used to analyze the function of the F. fujikuroi mep genes (24, 25).
Bacterial strains and plasmids.
Escherichia coli strain Top10 (Invitrogen, Groningen, The Netherlands) was used for plasmid propagation. Genomic DNA fragments carrying the F. fujikuroi mepA, mepB, and mepC genes or parts of the genes were cloned into the vector pUC19 (Fermentas, Germany). For the replacement of mepA, a 1.1-kb KpnI/SalI fragment of the 5' noncoding region and a 0.9-kb HindIII/BamHI fragment of the 3' noncoding region were cloned into the plasmid pUCH2-8 (1) carrying the hygromycin B resistance cassette. A KpnI/BamHI fragment of the resulting replacement vector, pmepA, carrying both flanks and the hygromycin resistance cassette, was used for gene replacement experiments. To construct the mepB gene replacement vector, a 0.8-kb SacII/XbaI fragment of the 5' noncoding region and a 0.5-kb ClaI/SalI fragment from the 3' noncoding region were cloned into the plasmid pNRI (28) carrying the nourseothricin resistance gene nat1. For targeted replacement of mepC, a 0.7-kb SacI/XbaI fragment of the 5' noncoding region and a 0.8-kb HindIII/SalI right flank were cloned into vector pNR1.
For complementation of the mepB mutant with the wild-type mepB copy, a 4.8-kb genomic SacI fragment was cloned into the vector pPUCH2-8 (1) carrying the hygromycin resistance cassette. The circular vector pmepB-hyg was used to transform the mepB mutant strain T1 (mepB-T1, where T1 is for transformant 1). For constructing the mepC overexpression vector, the promoter of the F. fujikuroi glnA gene (46) was amplified by PCR using the primers glnA-prom-XbaI and glnA-prom-Bam. The coding region of the mepC gene was amplified with the primers MepC-Bam-F and MepC-Hind-R. The restricted promoter and mepC fragments were cloned by one step into the XbaI/HindIII-restricted vector pUCH2-8.
Screening of genomic library. About 40,000 recombinant phages of the F. fujikuroi m567 genomic library (28) were plated with E. coli strain XII-Blue MRF' and screened by plaque hybridization as described previously (42). Plaque lifts (Gene Screen nylon membranes; DuPont, Germany) were hybridized with [32P]dCTP-labeled 0.5-kb PCR fragments of the F. fujikuroi mepA, mepB, and mepC genes. Hybridization and washing steps were performed at 65°C. The blots were washed at 65°C (once with 2 x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% sodium dodecyl sulfate [SDS]; once with 0.1 x SSC and 0.1% SDS) as described previously (46). Putative positive phages were purified in a second screening round. Phage DNA was isolated as described previously (42) and used for restriction analysis and subcloning.
DNA isolation. Lyophilized mycelium was ground into a fine powder with a mortar and pestle and dispersed (in the case of DNA for use in PCR) in extraction buffer as described by Cenis (5). DNA for Southern hybridization experiments was prepared following the protocol of Doyle and Doyle (9). Plasmid DNA was extracted using a plasmid extraction kit (Genomed, Germany).
Southern-blot analysis. For Southern blot analysis, genomic, plasmid, or phage DNA was digested to completion with appropriate restriction enzymes (Fermentas, Germany), fractionated in 1.0% (wt/vol) agarose gels, and transferred to nylon N+ membranes (Amersham, Germany) by vacuum blotting. DNA probes were randomly labeled, and hybridizations were carried out overnight at 65°C. The blots were washed under hybridization conditions (2 x SSC-0.1% SDS at 65°C followed by 0.1 x SSC-0.1% SDS).
Northern blot analysis. For RNA isolation, the fungal strains were grown in ICI medium with 20 mM glutamine as a nitrogen source instead of ammonium nitrate for 3 days on a rotary shaker at 28°C. After 24, 48, and 72 h, mycelia were harvested and used for RNA preparation. For shift experiments, the mycelium was grown for 3 days in ICI medium with 20 mM glutamine, washed, and transferred into synthetic ICI medium without nitrogen for 2 h to induce starvation; the mycelium was then shifted into medium without nitrogen or with 10 mM NH4NO3 or 10 mM glutamine, with (200 ng/ml) or without rapamycin (Calbiochem). Mycelia were harvested after 1 h. The irreversible inhibitor of the GS, L-methionine sulfoximine (MSX) (Sigma-Aldrich, Germany), was added in a concentration of 4.0 mM. Total F. fujikuroi RNA was isolated using an RNAgents total RNA isolation kit (Promega, Germany).
PCR and reverse transcription-PCR (RT-PCR).
PCR primers MepL 5'-CAATGGTTCTTCTGGGGCTACTC-3' and MepR 5'-CGAACCAGCCGA ACCAGAGGAA-3' were used for amplification of F. fujikuroi ammonium permease genes mepA and mepB. For cloning the third ammonium permease gene, mepC, primers MepC-F1 5'-CTTCAACATGTCTTATGTTATCCC-3' and MepC-R1 5'- GCAAGTTTAAGCATGCTTCTCG-3' were used. To construct gene replacement vectors pmepA, pmepB, and pmepC, the 3' and 5' noncoding regions were amplified with the following primers: epA-GR1-KpnI, 5'-GGAATTCGGTACCCTTGAGGAC-3'; MepA-GR2-SalI, 5'-TGTAAGGAGTGGTCATGTCGAGCAGACTGC-3'; MepA-GR3-Hind, 5'-TAAGCTTCGAGAAGAAGTGGAGCACC-3'; MepA-GR4-BamHI, 5'-CGGATCCTTGATAGGAGTATAAAGGACC-3'; MepB-GR1-SacII, 5'-CGAAAAAAACGTCACCGCGGTACCGG-3'; MepB-GR2-XbaI, 5'-CTAGAGGAACCATTAACAGCACTAGAGC-3'; MepB-GR3-ClaI, 5'-GATCGATCGATATCGATAACGAGTCCG-3'; MepB-GR4-SalI, 5'-CGTCGACTTCAGACTGCTGCTATTAGTCC-3'; MepC-GR1-SacI, 5'-TTGGGGAGCTCGTACTGATAAACATCCATGAGATGG-3'; MepC-GR2-XbaI, 5'-GCAATAAAATAACCCCTCTAGATCAAACAAGG-3'; MepC-GR3-HindIII, 5'-GGTGATTAACGAGAAGCATGCTTAAGCTTGC-3'; MepC-GR4-SalI, 5'-ATGTCGACGTTACTCTGGCTCCCAGATCAACACG-3'.
Derived fragments were first cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. For constructing the yeast expression vectors, the full-length cDNA fragments of the three F. fujikuroi mep genes were generated by RT-PCR using the following primers: MepA-Sca, 5'-GCAGTACTGTAAAAATGACCACTCC-3'; MepA-Xba, 5'-CACTAATCTAGACCTTTAGACC-3'; MepB-Sca, 5'-GCAGCCTAGTACTATCGCAAAAATGCTCTCG-3'; MepB-Xba, 5'-GGTATCTAGAACTCGACAATTCC-3'; MepC-ScaI, 5'-CCACGAGTACTTATAAACACAATAATCAAAAATGTC-3'; and MepC-Xba, 5'-GCAATAAAATAACCCCTCTAGATCAAACAAGG-3'.
The cDNA fragments of mepA, mepB, and mepC were first cloned into the PCR cloning vector pCR2.1-TOPO (Invitrogen) and then subcloned into the ScaI/XbaI-restricted yeast expression vector pYES2.1 (Invitrogen) to yield complementation vectors pYes2.1-mepA, pYes2.1-mepB, and pYes2.1-mepC. These plasmids were sequenced and used for complementation of the yeast triple mep1 mep2 mep3 and mep2 mutants. Vector pYes2.1-mepB was also used for complementing the F. fujikuroi mepB mutant.
For constructing the mepC overexpression vector, the following primers were used: MepC-Bam-F, 5'-ATGGATCCAACATGTCTTATGTTATCCCTGG-3'; MepC-Hind-R, 5'-ATAAGCTTCTTTGCGTGCTACTCCTAGCATCCCGC-3'; glnA-prom-Xba, 5'-AGTCTAGACGGAGCAAAGCGGTTTATATCCGCC-3'; and glnA-prom-Bam, 5'-TTGGATCCTGTGAATGTGGTTGTGATACGGGG-3'.
PCRs contained 25 ng of DNA, 50 ng of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 2 U of Taq polymerase (Red Taq; Sigma-Aldrich, Germany) in 50 µl. PCR was carried out at 94°C for 4 min followed by 30 cycles of 94°C for 1 min, 53 to 57°C for 30 s, and 72°C for 1.5 min. For RT-PCR 1 µg of total RNA of nitrogen-starved wild-type mycelium served as a template to create cDNA by using a One-step qRT-PCR kit (Invitrogen, Groningen, Germany).
Fungal transformations.
Preparation of protoplasts from F. fujikuroi mycelium was carried out as described previously (50). A total of 107 protoplasts of strain IMI58289 were transformed with 10 µg of the KpnI/BamHI, SacI/SalI, or SacI/SalI fragment of the replacement vectors pmepA, pmepB, or pmepC, respectively. For gene replacement, transformed protoplasts were regenerated at 28°C in a complete regeneration agar (0.7 M sucrose, 0.05% yeast extract, 0.1% Casamino Acids) containing 120 µg/ml hygromycin B (for pmepA) (Calbiochem, Germany) or 100 µg/ml nourseothricin (for pmepB and pmepC) (Werner-Bioagents, Germany) for 6 to 7 days. For construction of mepA mepB double mutants, strain mepA-T10 was transformed with 10 µg of the SacI/SalI-fragment of vector pmepB. For construction of mepB mepC double mutants, strain mepB-T1 was cotransformed with 10 µg of the SacI/SalI fragment of vector pmepC and 10 µg of the vector pUCH2-8 carrying the hygromycin resistance cassette. For complementation, the mepB-T1 mutant was cotransformed with 10 µg (each) of the hygromycin B resistance-mediating vector pUCH2-8 and the pYES2.1 vector carrying the mepB cDNA fragment. The transformants were additionally selected by regeneration on a medium containing low ammonium concentrations (1 mM ammonium citrate) as the only nitrogen source. Single conidial cultures were established from hygromycin B- or nourseothrecin-resistant transformants and used for DNA isolation and Southern blot analysis.
Analysis of mepA, mepB, and mepC function in S. cerevisiae.
To test the function of the F. fujikuroi mep genes in S. cerevisiae, the vectors pYes2.1-mepA, pYes2.1-mepB, and pYes-mepC and control vectors pYes2.1-Mep2 and pYes2.1 were transformed into the haploid S. cerevisiae strain MLY131 (mep1 mep2 mep3). The wild-type MLY40 containing pYes2.1 served as a control strain. Growth of transformants under ammonium-limiting conditions was assayed by plating serial dilutions of washed overnight cultures onto SD plates (yeast nitrogen base with ammonium sulfate and without amino acid supplements plus 2% glucose) and SLADG plates (yeast nitrogen base without ammonium sulfate and without amino acids supplemented with 50 µM ammonium sulfate, 2% galactose, and 0.2% glucose). Pseudohyphal growth was analyzed by streaking transformants of the diploid MLY108a/ (mep2/mep2) S. cerevisiae strain containing the F. fujikuroi pYes2.1-mep vectors to single cells onto SLADG agar, which were then grown for 6 days at 30°C and photographed.
DNA sequencing and sequence homology searches. DNA sequencing of recombinant plasmid clones was accomplished with the automatic sequencer Li-Cor 4000 (MWG, München, Germany). The two strands of overlapping subclones obtained from the genomic DNA clones were sequenced using the universal and the reverse primers or specific IRD-800-labeled oligonucleotides obtained from MWG Biotech (Munich, Germany). DNA and protein sequence alignments were done with DNA Star (Madison, WI). Sequence homology searches were performed using the NCBI database server. Protein homology was based on BlastX searches (2). For further investigations, the programs of DNA STAR Inc. (Madison, WI) were used.
[14C]methylamine uptake. The analysis of methylamine uptake was carried out as previously described (44). Briefly, the S. cerevisiae diploid mep1 mep2 mep3 null strain was transformed with the yeast vector pYES2.1 that contained mepA, mepB, and mepC under the control of the GAL1 promoter. Individual transformants were grown in synthetic medium lacking uracil with galactose (3%) as the carbon source and proline as the nitrogen source (1 mM) for 6 h. The cells were pelleted, washed, and resuspended in phosphate buffer (20 mM; pH 7) to an optical density at 595 nm of 8 and incubated on ice. Aliquots were added to phosphate buffer (20 mM; pH 7) containing 0.1 mCi [14C]methylamine hydrochloride (MP Biomedicals, Inc) and increasing concentrations of methylamine. The cells were resuspended in a water bath at 30°C, and samples of 1 ml were removed at 1-min intervals and washed over GC Whatman filters. The level of [14C]methylamine was then quantified using liquid scintillation counting.
GA determination by TLC. Amounts of produced GA3 and GA4/7 were determined by thin-layer chromatography (TLC) on silica gel eluted with ethyl acetate-chloroform-acetic acid (60:40:5).
Nucleotide sequence accession numbers. The sequences of the F. fujikuroi mepA, mepB, and mepC genes were deposited in the GenBank database under accession numbers AM168272, AM168273, and AM283470, respectively.
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RESULTS |
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All three mep genes are strongly repressed by ammonium and glutamine and derepressed under nitrogen starvation conditions (Fig. 3). The three mep genes were not expressed in the areA deletion strain under all of the growth conditions tested, and several single and double GATA or TATC sequence elements were found in the 5' noncoding regions of all three genes (data not shown), which is consistent with the idea that the mep genes are directly regulated by AreA. Interestingly, the mepA and mepB genes are also not expressed in the glnA mutant whereas mepC is upregulated in this mutant except for the medium with glutamine (Fig. 3).
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) in which all three Mep-encoding genes had been deleted. All three F. fujikuroi mep genes were able to complement the yeast mep1 mep2 mep3 triple mutant to restore growth on low-ammonium medium, consistent with these encoding functional ammonium permeases (Fig. 4). The level of growth was equal to the same strain transformed with the S. cerevisiae MEP2 gene that was expressed from the same vector as the F. fujikuroi mep genes (Fig. 4).
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/mep2 strain. Transformants containing the individual F. fujikuroi genes were grown on low-ammonium medium for 6 days. The mepA and mepC permease genes were able to complement the mep2/mep2 mutant under these conditions, with MepA being the stronger inducer of pseudohyphal growth. The F. fujikuroi mepB gene did not complement the S. cerevisiae mutant, notwithstanding the fact that it was expressed from the same promoter as the other two permease genes. [14C]methylamine uptake. To confirm that the F. fujikuroi mep genes encode functional ammonium permeases, we assayed their ability to mediate [14C]methylamine uptake when expressed in a S. cerevisiae Mep-deficient mutant from a galactose-induced promoter. Cells were grown to mid-log phase using proline as a nitrogen source, and the rate of methylamine uptake over a range of methylamine concentrations was determined. All three F. fujikuroi Mep proteins mediated detectable methylamine uptake that increased with increasing concentrations of external methylamine. In the case of MepA and MepC, the permeases became saturated over the range of methylamine concentrations used (Fig. 5). MepA has the highest relative affinity for methylamine with an apparent Km of 140 µM. MepC exhibited a lower affinity for methylamine with an apparent Km of 1.37 mM and a higher Vmax than MepA. Under these experimental conditions, the highest external concentrations of methylamine did not saturate MepB and precluded the determination of the kinetic parameters of this particular permease. We note that it was also not possible to determine the Km of the low-affinity permease Ump1 from U. maydis using the same experimental system (44). We can conclude, therefore, that MepB has the lowest affinity and highest capacity for methylamine of the three F. fujikuroi ammonium permeases.
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mepA (hygromycin resistance marker), pmepB, and pmepC (both with the nourseothricin resistance cassette), respectively (Fig. 6) (see Materials and Methods). Altogether, three mepA mutants (mepA-T4, -T9, and -T10), three mepB mutants (mepB-T1, -T2, and -T10) and four mepC mutants (mepC-T4, -T11, -T17, and -T22) were obtained. Deletion mutants transformed with the same replacement cassette showed similar phenotypes whereas transformants with ectopic integrations of the replacement cassette behave as the wild type.
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mepA strain exhibited wild-type levels of growth on all ammonium concentrations. This is consistent with our data from [14C]methylamine uptake experiments showing that this permease seems to have a minor role in ammonium acquisition not only in S. cerevisiae but also in F. fujikuroi and that the lack of MepA is fully compensated by MepB and MepC. The mepB strain, and to a lesser extent the mepC strain, exhibited a decreased growth rate on medium with decreasing levels of ammonium, which was most severe on plates that completely lacked ammonium. Under these latter conditions, the mepA and mepC strains were still able to form a thin mycelium, similar to that on nutrient-free water agar, in contrast to the mepB strains. (Fig. 7A). This is consistent with the idea that MepB is the major ammonium transporter in F. fujikuroi under these growth conditions, with the elimination of this permease causing a strong starvation-induced growth inhibition, probably due to a significant defect in ammonium transport. Surprisingly, 100 mM ammonium revealed a rather inhibitory effect on growth of the wild type and the mutants; the reasons for this growth inhibition are not yet clear.
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mepA mepB and mepB mepC double mutants. Both the mepA mepB and the mepB mepC strains exhibited no further reduction in growth rate under low-ammonium conditions compared with the mepB single mutant. The lack of increased sensitivity to low-ammonium growth by the additional deletion of either mepA or mepC confirmed the major role of MepB. The minimal growth of the mepB mepC strain under these conditions is probably attributed to nonspecific ammonium uptake and the presence of MepA. We were not able to construct a triple mepA mepB mepC mutant due to the lack of a suitable third resistance marker for F. fujikuroi. Therefore, the extent of nonspecific ammonium uptake cannot be precisely determined.
To quantify the growth defect of the mutants lacking one or two Mep permeases, we grew the wild type and the single and double mutants in liquid synthetic medium with ammonium sulfate (10 mM) or glutamine (10 mM) for 48 h and determined the dry weight of the mycelia. While all strains produce almost the same biomass when grown with glutamine as the nitrogen source, the mepB strain and mepA mepB and mepB mepC double mutants produced significantly fewer mycelia when grown on ammonium (Fig. 7B). To link the phenotype of the mepB mutants with the deletion of the mepB permease gene, we complemented the mutant mepB-T10 with the mepB wild-type copy cloned into the vector pGPC1. Hygromycin-resistant transformants were screened for their ability to grow on low-ammonium-containing medium (1 mM ammonium citrate) and for the integration of the complementing mepB gene. Five of the 12 transformants tested revealed the expected PCR fragment for the mepB gene, and these transformants exhibited almost the same growth as the wild type and showed wild-type-like expression of the mepB gene (data not shown). Thus, the growth defect of mepB mutants on low ammonium is clearly linked to the deletion of the mepB ammonium permease gene.
Deletion of mepB but not mepA or mepC confers highly deregulated secondary metabolism. To date, the impact of single ammonium transporters on secondary metabolism has not been studied in filamentous fungi. In F. fujikuroi, two different nitrogen-free metabolites, the GAs and the red pigment bikaverin, are strictly regulated by nitrogen metabolite repression. To test if the F. fujikuroi ammonium permeases influence GA and bikaverin biosynthesis, we analyzed the production of these secondary metabolites in the mep deletion strains. In wild-type cells, the initiation of bikaverin biosynthesis is a marker for nitrogen depletion, and the production of the pink pigment is accompanied by induction of GA biosynthesis (23, 50).
Wild-type and mutant strains were grown for 48 h in a medium containing 10 mM glutamine to support equal growth for all strains. Aliquots were inoculated into synthetic ICI medium containing 20 mM ammonium or glutamine. The color of the cultures was monitored at 15, 24, 48, and 72 h. The color of the wild-type and mepA cultures changed from white (up to 24 h) to light pink (30 h to 48 h) to yellowish pink (72 h) (Fig. 8). In contrast, the single mepB and the double mepA mepB and mepB mepC mutant strains displayed a red pigmentation already 15 h after inoculation that increased with longer incubation (Fig. 8). The pigmentation of the mepC deletion strain was also apparent after 15 h of cultivation but was not as intense as in strains that lacked mepB. Surprisingly, a slightly earlier pigmentation of mepB, mepA mepB, and mepC mutants was also observed in cultures with glutamine as nitrogen source (Fig. 8).
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mepA and mepC mutants behaved as the wild type: these strains did not produce visible amounts of GAs at 30 and 48 h. However, the mepB strain and the mepA mepB and mepB mepC double mutants had already initiated GA3 production by 30 h (Fig. 8). With glutamine, all strains started to produce GA3 only at 72 h (Fig. 8). Thus, deletion of mepB affects the biosynthesis of both secondary metabolites in a similar fashion. The early bikaverin and GA formation in the medium containing ammonium confirms the importance of MepB in ammonium uptake and cellular nitrogen homeostasis. Gene expression of secondary metabolism genes is deregulated in mepB deletion strains. We analyzed the expression of GA and bikaverin biosynthesis genes using the same cultivation conditions that had been used for monitoring bikaverin and GA production. In medium containing 20 mM ammonium, the bikaverin biosynthetic genes pks4, MT, and MO (encoding the polyketide synthase, a methyltransferase, and a monooxygenase from the bikaverin gene cluster) (23; also P. Wiemann and B. Tudzynski, unpublished data) and the GA biosynthetic genes (cps/ks and P450-1) (reviewed in reference 51) were expressed by 24 h in the mepB single and double mutants. The same transcripts were detectable only at 48 or 72 h in the wild-type and the mepA and mepC mutant strains (Fig. 9). This pattern of expression has also been observed for other areA target genes, e.g., the peptide transporter gene mtd1 and the amino acid permease gene aap8 (B. Schönig and B. Tudzynski, unpublished data; also data not shown) as well as a set of GS target genes, e.g., ddr48 and cipC (Fig. 9), which are not expressed in the glnA mutant (46). As expected, nitrogen deregulation is most significant when the cultures contain ammonium as the nitrogen source. However, the expression of the GA and bikaverin biosynthetic genes is also partially deregulated in single and double mepB mutants and, for some genes, also in mepC mutants with glutamine as a nitrogen source, although the effect is not as clear as with ammonium (Fig. 9).
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mepB recipient strain (Fig. 11A). Statistical analysis of the growth rate of the mepC-overexpressing strains compared with that of the mepB mutant and the wild type is shown in Table 2.
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mepB+mepC-1, -2, -3, and -10) in ICI medium with 20 mM ammonium sulfate. Surprisingly, the mepC-overexpressing mutants already showed a strong pigmentation after 15 h, similar to the mepB mutant despite having a wild-type growth pattern (Fig. 11B). The mycelia were harvested after 24 h and used for expression studies by Northern blot analysis. The mepC gene was strongly up-regulated in the mutants, as expected. The GA and bikaverin genes are highly expressed at 24 h as is the mepB mutant, despite the almost full restoration of growth (Fig. 11C). These data suggest that MepB plays an additional role in nitrogen regulation in F. fujikuroi, probably by sensing and transducing the signal of ammonium availability. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, the MepB permease of F. fujikuroi does not group together with MeaA or yeast Mep1, despite their functional similarity as high-capacity permeases. Instead, MepB groups with the A. nidulans and Tuber borchii high-affinity permeases MepA and TbAMT1, respectively (34, 37). MeaA shows high sequence similarity to the F. fujikuroi MepC permease, which is also a high-capacity permease according to the reduced growth on medium containing 1 to 100 mM ammonium. In contrast to mepB and mepC mutants, mepA mutants exhibit no obvious phenotype. They grow at the same rate as the wild type and produce colonies with thin mycelia, even on plates without nitrogen. Analysis of methylamine uptake confirmed that MepA is a high-affinity and low-capacity permease. MepA groups into an AmtB/Mep subclass together with the A. nidulans MepA and the F. fujikuroi high-capacity permease MepB. Therefore, the two F. fujikuroi permeases MepA and MepB are phylogenetically highly related but differ in their affinity for ammonium and their transport capacity, demonstrating that a high degree of sequence similarity cannot be strictly correlated with functional inferences.
The expression of mepA, mepB, and mepC is highly dependent on AreA.
The expression of all three mep genes is uniformly regulated by AreA. In the areA mutant, no transcript levels were detected for mepA, mepB, and mepC, consistent with the finding that the F. fujikuroi areA mutant does not grow on medium with ammonium as the only nitrogen source. This contrasts with A. nidulans where disruption of the zinc finger region of areA does not result in loss of ability to grow on ammonium but prevents utilization of nitrogen sources other than ammonium and glutamine (21).
The expression of several Mep2-type permeases, such as the S. cerevisiae MEP genes (30) and the four permeases in A. nidulans (34, 35, 36), is nitrogen regulated and depends on activation by the transcription factors Gln3 and AreA, respectively. Genes encoding permeases of the Mep1/Mep3 type, such as Mep1 in C. albicans (3) and MeaA in A. nidulans (34, 35), are expressed at much lower levels than the high-affinity Mep2-type transporters. In F. fujikuroi, mepA shows the highest expression level similar to other Mep2-like permeases. Interestingly, the expression of mepC is upregulated in the glnA mutant in medium without nitrogen or with 10 mM ammonium, whereas glutamine overcomes this effect. In contrast, the mepA and mepB genes are repressed in both the areA and glnA mutants. Further studies will be required to identify the mechanism of GS-dependent expression of the mep genes.
Does MepB participate in nitrogen regulation? Some fungal ammonium permeases have evolved a regulatory function. In S. cerevisiae, several plasma membrane-localized permeases are able to sense the environmental availability of amino acids, ammonium, and sugars (10, 52). Among the three Mep proteins, Mep2 is a receptor for ammonium and is required for pseudohyphal growth under nitrogen starvation conditions (25). In C. albicans, CaMep2, but not CaMep1, also induces filamentous growth under limiting nitrogen conditions via the activation of both mitogen-activated protein kinase and the Ras1-cyclic AMP-dependent pathways (3). The maize pathogen U. maydis expresses two ammonium permease-encoding genes, ump1 and ump2 (44). Ump2 is required to trigger filamentous growth on low-nitrogen medium and complements the pseudohyphal growth defect of the S. cerevisae mep2 mutant, consistent with a sensor role (44). Similarly, it has been proposed that in the mycorrhiza fungus H. cylindrosporum, the high-affinity ammonium transporters Amt1 and Amt2 induce, via an as yet unknown signal transduction cascade, a switch in the fungal growth mode during the formation of mycorrhiza (17).
The molecular mechanisms involved in transporter-mediated ammonium regulation are not presently known although some important aspects have been identified in yeast that may be conserved within fungi. The Mep2-like permeases are high-affinity transporters and are the most highly transcribed in response to ammonium limitation. Promoter exchange and reporter gene experiments show that CaMep2 needs to be expressed at very high levels to mediate filamentous growth induction under low-ammonium conditions (3). A mutant strain lacking such a regulatory permease would also be expected to exhibit a signaling phenotype, and no phenotype that could be attributed to a reduction in nitrogen availability, such as a reduction in growth rate. Other characteristics of the yeast ammonium permeases are less predictive with regard to a role as regulatory proteins. Sequence alignments of the currently available Meps in the database show that fungal Meps contain a putative protein kinase A (PKA) phosphorylation site [RRX(S/T), where X is any residue] that is not present in those from other organisms (44). Point mutations in these sites in Ump2 (Ser288-Ala) and Mep2 (Thr288-Ala) do not abolish ammonium transport, but both mutant Meps fail to complement the pseudohyphal growth defect of the S. cerevisiae mep2 mutant (44). However, nonsensing homologues of Mep2 (e.g., Mep1 and Mep3 of S. cerevisiae) also contain a similar potential PKA phosphorylation site. In addition, the S. cerevisiae Mep2 is glycosylated at the fourth amino acid. However, Mep2N4Q mutation of the S. cerevisiae glycosylation site does not prevent pseudohyphal growth (32). Many of the Mep2-specific features, such as glycosylation and putative PKA phosphorylation sites, are found in all three F. fujikuroi Mep proteins. In addition, all Fusarium Meps are predicted to have the same overall structure as Mep2, including the putative sensing loop (25). It is therefore challenging to identify conserved characteristics within the ammonium permeases of filamentous fungi that define a regulatory function.
The data presented in this study are most consistent with MepA having the characteristics of a regulatory permease: the gene is highly expressed, the mepA mutants show no severe growth defect on low ammonium, MepA (but also MepB and MepC) is predicted to be glycosylated, and MepA is a high-affinity transporter that is able to induce pseudohyphal growth in a mep2-deficient S. cerevisiae mutant. MepC also restores the pseudohyphal growth in the yeast mep2 mutant, but the deletion of mepC conferred an obvious growth phenotype in F. fujikuroi. However, the expression patterns of the AreA target genes argue against a role of MepA or MepC as extracellular ammonium sensors that link ammonium availability to the regulation of AreA activity. If MepA or MepC were a sensor permease, then the signal for nitrogen sufficiency would be transduced in mepB mutants, resulting in strong repression of AreA target genes, but this is not the case.
In contrast, MepB does not restore pseudohyphal growth of the S. cerevisiae mep2 mutant, and mepB mutants do not grow under low-ammonium conditions, making it appear to be a less likely candidate for a regulatory permease. However, MepB is the only permease affecting the regulation of AreA target genes, such as the GA and bikaverin biosynthesis genes, which are derepressed in the mepB mutant under ammonium-sufficient conditions. In addition, a partial derepression of GA and bikaverin genes in the mepB mutants was observed also on glutamine, similar to C. albicans where the Mep2 orthologue is required for nitrogen limitation-induced filamentation under conditions of both ammonium and amino acid limitation (3).
There are two possible explanations for the strong impact of MepB on regulation. First, the effects on gene expression may be due to reduced ammonium transport, in agreement with the growth defect of the mepB mutant and with methylamine uptake experiments demonstrating that MepB is the major ammonium transporter in F. fujikuroi. Second, MepB might play an additional role in nitrogen regulation despite not exhibiting a regulatory function in the heterologous S. cerevisiae model. If the derepressing effect on nitrogen-regulated genes in the mepB mutant is the result of ammonium limitation due to the loss of the major transporter, overexpression of the second high-capacity permease, MepC, should restore not only the wild-type growth but also the wild-type expression levels of these genes. However, despite the significantly increased growth of the mepC-overexpressing mutants (Fig. 11), the deregulation of GA and bikaverin biosynthesis genes as well as other AreA-dependent genes is the same as in the mepB mutant. These data support our suggestion that MepB might play an important role in transport and sensing of ammonium although this permease does not complement the pseudohyphal growth defect in yeast. However, due to the evolutionary distance between F. fujikuroi and S. cerevisiae, heterologous expression of F. fujikuroi mep genes need not necessarily mimic all of the regulatory events or expression levels that occur in the natural host.
However, more definitive evidence will be required before a sensing function can be attributed to any of the F. fujikuroi ammonium permeases. Such evidence includes the isolation of mutants, such as those recently identified in Mep2 of S. cerevisiae (J. Rutherford et al., personal communication), that separate the transport and sensing function of the permease, a demonstration of interactions with signal transduction proteins, or the identification of downstream physiological events such as altered transcriptional programs. This will also include the identification of mutants analogous to the hyperactive Mep2G349C mutant of S. cerevisiae that restores dimorphic growth in a pseudohyphal-deficient mutant (npr1) and thereby establishes a clear correlation between Mep2 dependent transport and signaling (4).
Is the metabolism of ammonium essential for triggering nitrogen repression? In S. cerevisiae, the stimulus for the expression of nitrogen-regulated genes by the AreA homologue Gln3 is a drop in the intracellular level of glutamine (7, 27). Therefore, we sought to establish whether the reduced intracellular glutamine level, resulting from the compromised ammonium transport capacity in the mepB mutants, or the decreased ammonium availability itself triggers the activation of AreA via a yet unknown signaling cascade and the derepression of AreA target genes. To answer this question, we inactivated both the GdhA and GS pathways for ammonium incorporation into metabolism. For this, we used a gdhA deletion mutant and treated the mycelium with the specific GS inhibitor MSX. Ammonium-induced strong repression of GA and bikaverin biosynthetic genes was not prevented under these conditions, demonstrating that the intracellular level of glutamine is not the only signal sensed by the fungal cells that activates/inactivates signaling pathways to AreA and that ammonium itself is an effector of nitrogen metabolite repression in F. fujikuroi.
Conclusions. We identified one of the three ammonium permeases, MepB, as the major transporter and as a potential element in the F. fujikuroi nitrogen regulation network. Although the inability of MepB to restore the pseudohyphal growth defect of S. cerevisiae mep2 mutants argues against a direct role of MepB as a regulator, at least in a heterologous organism, several other features support such a role. The strongest argument for a regulatory role is the overexpression of mepC encoding the second high-capacity transporter in F. fujikuroi, which resulted in significantly improved growth but did not prevent the deregulated expression pattern of the nitrogen-regulated secondary metabolism genes.
Recently, we showed that GA and bikaverin biosynthetic genes are strongly repressed in a glnA mutant even under nitrogen-limiting conditions, despite the fact that the intracellular glutamine pool is drastically reduced. These results are in contrast to our expectation and suggest a regulatory role of GS in addition to its enzymatic function, e.g., by protein-protein interactions with other components of the nitrogen regulation network (46). Summarizing our current knowledge of the role of GS and other components involved in nitrogen regulation of secondary metabolism in F. fujikuroi and the postulated regulatory role of MepB, we propose a model for nitrogen sensing and signaling (Fig. 12). In this model, MepB is the major ammonium transporter which is able to sense ammonium (nitrogen) availability and to transduce a repressing signal to AreA by an as yet unknown signaling pathway. Under ammonium limitation conditions, GS binds to MepB, thereby blocking the transduction of this repressing signal and resulting in the induced expression of glnA and probably increased GS levels. The missing signal leads to an activation of AreA and derepression of AreA target genes, such as the mep and the GA and bikaverin biosynthetic genes. This model can also explain why GS is needed for the expression of the GA and bikaverin biosynthesis genes under nitrogen starvation conditions (46). The inability of GS to bind to MepB in the glnA deletion mutant would enable the putative sensor MepB to constitutively activate signaling, resulting in strong repression of AreA target genes. In support of this model, a close metabolic coupling of the membrane-bound GS to the ammonium channel AmtB has been recently shown for E. coli (19). This model will provide the basis of future work to identify the molecular mechanisms that link nitrogen availability to the regulation of secondary metabolism in F. fujikuroi.
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ACKNOWLEDGMENTS |
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We thank Sabine Huber (University Münster) for excellent technical assistance and Sabrina Wemhoff for cloning and sequencing the F. fujikuroi mepC gene. We also thank Mike Perlin for supplying us with the pYes2.1-Mep2 plasmid.
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FOOTNOTES |
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