ABSTRACT
Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3′ end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism.
When cultured under ligninolytic (idiophasic) conditions, the lignin-degrading fungus Phanerochaete chrysosporium produces two families of peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP), along with an H2O2-generating system (18, 20, 26, 27). MnPs are heme-containing, H2O2-dependent, extracellular glycoproteins with molecular masses ranging from 45 to 49 kDa (15, 20, 39). MnP oxidizes Mn2+ to Mn3+ and the latter, chelated with oxalate or another dicarboxylic acid, acts as a diffusible mediator to oxidize polymeric lignin, lignin model compounds, and aromatic pollutants (14, 15, 35, 41, 42, 44, 48). Virtually all white-rot basidiomycetous fungi produce MnP (22, 37, 38). In P. chrysosporium, MnPs are secreted as a series of isozymes, encoded by a family of related genes (18, 20). Over the past 15 years, cDNA and genomic sequences of three MnP-encoding genes (mnps) from P. chrysosporium, mnp1, mnp2, and mnp3, have been characterized (3, 17, 32, 36, 39).
Both MnP and LiP from P. chrysosporium are idiophasic proteins, the production of which is triggered by the depletion of nutrient nitrogen (17, 18, 29, 39, 46). Furthermore, MnP production is uniquely dependent on the presence of sufficient Mn2+ in culture (7, 8, 16). Northern blot and reverse transcription-PCR analyses have shown that both nitrogen limitation and Mn2+ supplementation are required to activate the transcription of MnP-encoding genes (7, 8, 12, 13). When P. chrysosporium is grown in nitrogen-limited cultures containing 180 μM Mn2+, the mnp transcript is first detected on day 5 following inoculation. In contrast, no mnp transcript is observed in nitrogen-sufficient cultures or in nitrogen-limited cultures without exogenous Mn2+ (8, 12, 16). The addition of Mn2+ to 5-day-old nitrogen-limited, Mn2+-deficient cultures results in detectable mnp transcript within 40 min, and the amount of mnp transcript is a function of the Mn2+ concentration, up to a maximum of 180 μM (7, 32).
Several short sequences in the promoter regions of P. chrysosporium mnps conform to conserved cis-acting promoter elements, including inverted CCAAT boxes, metal response elements (MREs), heat shock elements, and a binding site for activator protein 2 (AP-2) (10, 17, 18, 20). However, the involvement of these sequences in the regulation of mnp gene expression has not been demonstrated.
Other than the studies described above, very little is known about Mn2+-dependent regulation of gene expression in fungi. In contrast, gene expression regulation by Mn2+ is much better studied in bacterial systems. Perhaps the best-understood system occurs in Bacillus subtilis, where the Mn2+-responsive metalloregulatory protein MntR appears to repress the transcription of several genes encoding Mn2+ transporters (mntH and mntABCD) in the presence of high levels of Mn2+. Interestingly, MntR also acts as a positive regulator for the mntABCD operon under low-Mn2+ conditions (40). Biochemical studies have demonstrated that the binding of MntR to the regulatory region of mntH requires Mn2+, while the interaction with the regulatory region of mntABCD does not depend on Mn2+ (30, 40).
To study the transcriptional regulation of mnp gene expression in P. chrysosporium, we recently developed an efficient reporter system based on the enhanced green fluorescent protein (EGFP) gene (egfp) (31). Efficient expression of egfp in P. chrysosporium is observed only when an intron is inserted at the 5′ end of the coding region of this gene. Based on a previously reported plasmid construct, pUMiGM3′ (31), which contains an expression cassette including the 1,528-bp mnp1 promoter (GenBank accession number AY566235 ) followed by an intron-containing egfp and the 250-bp mnp1 3′ untranslated region (UTR) (17), we have constructed a series of deletion, replacement, and translocation mutations in the mnp1 promoter. The effects of these mutations on Mn2+ dependence of the mnp1 promoter-directed egfp expression were measured. Herein, we report the characterization of a 33-bp sequence residing 521 bp upstream of the translation start codon in the mnp1 promoter that is, at least partially, responsible for Mn2+-dependent regulation of mnp1 promoter activity.
MATERIALS AND METHODS
Organisms. P. chrysosporium OGC101 and the auxotrophic Ura11 strain were maintained as described previously (1, 6). Escherichia coli strain DH5α was used for subcloning plasmids.
Construction of pUMiGM3′-A, pUMiGM3′-K, pUMiGM3′-E, and pUMiGM3′-ΔEK.The expression cassette of pUMiGM3′ (31), consisting of 1,528 bp of the mnp1 promoter (GenBank accession number AY566235 ), a 70-bp sequence that includes the first intron from the P. chrysosporium gpd gene, the egfp coding region, and 250 bp of the mnp1 3′ UTR (17), was subcloned into pUB, a vector containing the Schizophyllum commune ura1 gene as a selectable marker for transformation of the P. chrysosporium Ura11 auxotrophic strain (1) (Fig. 1). The mnp1 promoter contained unique AvrII, KpnI, and EagI restriction sites at −80, −521, and −774 bp positions with respect to the translation start codon (Fig. 1 and 2). Double digests of pUMiGM3′ with SphI/AvrII, SphI/KpnI, SphI/EagI, and EagI/KpnI released 1,448-, 1,007-, 750-, and 253-bp fragments of the mnp1 promoter, respectively. Each double digest was fractionated on a 1% agarose gel, and the egfp-containing fragments were purified using a gel extraction kit (QIAGEN). The remaining plasmids were blunt ended with T4 DNA polymerase (New England Biolabs) and religated with T4 DNA ligase (New England Biolabs) to yield pUMiGM3′-A, pUMiGM3′-K, pUMiGM3′-E, and pUMiGM3′-ΔEK, respectively (Fig. 2 and 3).
Restriction map of pUMiGM3′. The locations of the P. chrysosporium mnp1 promoter, the intron-containing egfp gene, the mnp1 3′ UTR, and the genes ampR and ura1 for selecting transformants in E. coli and in P. chrysosporium, respectively, are indicated.
GFP expression in transformants carrying pUMiGM3′, pUMiGM3′-K, and pUMiGM3′-E. The upper panel shows the deleted mnp1 promoter sequence (dashed line) in these pUMiGM3′ derivatives, as well as in pUMiGM3′-A. The lower panel shows the average fluorescence intensity of the two most fluorescent transformants carrying each of the three plasmids in 7-day-old HCLN (2% glucose, 1.2 mM ammonium tartrate, and Kirk's salts) cultures with and without exogenous Mn2+ (+Mn and −Mn, respectively). The average fluorescence intensity per milligram of total protein was determined from triplicate −Mn and +Mn cultures of each transformant, after deduction of the background fluorescence intensity per milligram of total protein in transformants carrying the blank vector pUB.
GFP expression in transformants carrying pUMiGM3′Δ-EK, pUMiGM3′ΔE197, pUMiGM3′ΔK(−48), and pUMiGM3′Δ48A. (A) The mutated mnp1 promoter sequences in various pUMiGM3′ derivatives. Dashed line, deleted sequence; black box, replaced sequence. (B) Nucleotide sequence of the 48-bp fragment immediately upstream of the KpnI site in the mnp1 promoter and in the synthesized 48-bp fragment in pUMiGM3′Δ48A that replaced the original 48-bp promoter fragment. The two identical sequences, conforming to a consensus AP-2 binding site, are in bold. The KpnI site is underlined. (C) Fluorescence intensities of the three most fluorescent transformants carrying each construct. Data were calculated as described in the legend to Fig. 2.
Construction of pUMiGM3′ΔE197.The construct pUMiGM3′ΔE197 contained a deletion of the 197-bp fragment immediately downstream of the EagI site (from position −774 to −578) in the mnp1 promoter. To create this construct, a forward primer that hybridized with the mnp1 promoter sequence from position −577 to −557 and contained an introduced EagI site at its 5′ end was synthesized. The reverse primer corresponded to the junction of the 3′ end of the mnp1 promoter and the 5′ end of the intron-containing egfp. With pUMiGM3′ as the template, a 0.6-kb fragment was amplified by PCR, digested with EagI and AvrII, and ligated into EagI/AvrII-digested pUMiGM3′, yielding pUMiGM3′ΔE197 (Fig. 3A).
Construction of pUMiGM3′ΔK(−48).The construct pUMiGM3′ΔK(−48) contained a 48-bp deletion immediately upstream of the KpnI site (from position −569 to −522) in the mnp1 promoter. A forward primer, hybridizing with the mnp1 promoter sequence from position −1,010 to −990, and a reverse primer, complementing the mnp1 promoter sequence from position −589 to −570 and containing an introduced KpnI site at its 5′ end, were prepared. With pUMiGM3′ as the template, a 0.5-kb fragment was amplified, digested with EagI and KpnI, and used to replace the EagI-KpnI fragment in pUMiGM3′, yielding pUMiGM3′ΔK(−48) (Fig. 3A).
Construction of pUMiGM3′Δ48A.The construct pUMiGM3′Δ48A contained a 48-bp fragment, with the sequence of the 5′ end of mnp1 exon 6 (17), inserted into the KpnI site of pUMiGM3′ΔK(−48). A pair of complementary 48-mer oligonucleotides, Oligo 1 (5′-CATTCACGTTCGACACGCAGGTGTTCCTCGAGGTGCTGCTCAAGGTAC-3′) and Oligo 2 (5′-CTTGAGCAGCACCTCGAGGAACACCTGCGTGTCGAACGTGAATGGTAC-3′), containing KpnI ends, was phosphorylated with T4 polynucleotide kinase (New England Biolabs), denatured by heating at 85°C for 5 min, and annealed by slowly cooling to room temperature. The resultant double-stranded DNA was ligated with KpnI-digested pUMiGM3′ΔK (−48) to yield pUMiGM3′Δ48A (Fig. 3A and B).
Construction of pUMiGM3′-48N.The pUMiGM3′-48N plasmid was constructed from pUMiGM3′ΔK (−48) by inserting the deleted 48-bp mnp1 promoter sequence into the unique NsiI site at position −398 in the mnp1 promoter. Oligonucleotides corresponding to each strand of the 48-bp mnp1 promoter sequence and containing NsiI ends were synthesized, combined, annealed, and ligated into NsiI-digested pUMiGM3′ΔK (−48) to yield pUMiGM3′-48N (see Fig. 5, below).
Construction of pUMiGM3′Δ24.The construct pUMiGM3′Δ24 contained a 24-bp deletion of the mnp1 promoter sequence from position −569 to −546. This deletion was located 24 bp upstream of the KpnI site and was constructed using the Transformer site-directed mutagenesis kit (Clontech Laboratories). A mutagenic primer, containing the deletion, and a selection primer, designed to change a unique SphI site located at the 5′ end of the mnp1 promoter into a NheI site, were synthesized. The two primers were used to synthesize a mutated strand of pUMiGM3′, following the manufacturer's instructions. pUMiGM3′Δ24 was recovered after transformation of E. coli (see Fig. 6, below).
Construction of pUMiGM3′Δ33.The construct pUMiGM3′Δ33 contained a 33-bp deletion immediately upstream of the KpnI site in the mnp1 promoter (from position −554 to −522). A forward primer, hybridizing with the mnp1 promoter sequence from position −1,010 to −990, and a reverse primer, complementing the mnp1 promoter sequence from position −574 to −555 and containing an introduced KpnI site at its 5′ end, were synthesized. With pUMiGM3′ as the template, a 0.5-kb fragment was amplified by PCR. The fragment was digested with EagI and KpnI and used to replace the EagI-KpnI fragment of pUMiGM3′ (see Fig. 6).
Fungal transformations.Plasmid DNA (∼1 μg), linearized with EcoRI, was used to transform protoplasts (2 × 106) prepared from basidiospores of the P. chrysosporium Ura11 strain (1) as described previously (4, 5). Approximately 100 Ura+ transformant colonies were obtained from each transformation. Among these colonies 40 were isolated from each transformation, and their ability to grow on minimal medium without uracil was verified. Transformants were purified by fruiting and basidiospore isolation as described previously (4, 5).
Culture conditions.Transformants were grown from conidial inocula at 37°C in stationary cultures containing 20 ml of high-carbon, low-nitrogen medium (HCLN; 2% glucose, 1.2 mM ammonium tartrate, and Kirk's salts) (28) supplemented with 180 μM MnSO4 in 250-ml Erlenmeyer flasks. Cultures were incubated under air for 4 days and then purged once with 100% O2. Seven-day-old cultures were harvested, and the intracellular GFP fluorescence was determined as previously described (31). Transformants that exhibited significant GFP fluorescence were grown in HCLN medium, with and without 180 μM MnSO4, and in high-carbon, high-nitrogen (HCHN; 2% glucose, 12 mM ammonium tartrate, and Kirk's salts) supplemented with 180 μM MnSO4, under the conditions described above and previously (31).
Intracellular GFP determinations.Approximately 100 mg of filtered damp mycelia was homogenized with glass beads (1 g) in a minibead beater (Biospec) in 1.5 ml of buffer (10 mM Tris-Cl [pH 8.0], 10 mM EDTA, 0.002% NaN3). The mixture was centrifuged at 10,000 × g at 4°C for 30 min. The total protein concentration of the cell extract was determined by the bicinchoninic acid method (Pierce) (43). The GFP fluorescence of 1 ml of supernatant was determined using an SLM Aminco 8000C spectrofluorometer with an excitation wavelength at 488 nm and an emission wavelength at 510 nm (31). Purified recombinant GFP (10 μg; Clontech Laboratories) was used to calibrate the spectrofluorometer at an arbitrary reading of 1,000 fluorescence units.
RNA extraction and Northern blotting.Total RNA of P. chrysosporium transformants was extracted from 5-day-old HCLN cultures with or without 180 μM MnSO4, as described previously (8). After spectrophotometric quantitation at 260 nm, the total RNA (20 μg per lane) was denatured and electrophoresed in a denaturing (0.6 M formaldehyde, 1% agarose) gel. Northern blotting was performed as described previously (8, 29). The coding region of egfp, the cDNA of P. chrysosporium mnp1, and the P. chrysosporium gpd gene were used as templates for random-primed synthesis of [α-32P]dCTP-labeled probes, using a Multiprime DNA labeling kit (Amersham). Probed RNA blots were washed and exposed to XAR-5 X-ray film (Kodak).
Southern blotting.Genomic DNA from various transformants was extracted as described previously (4). For Southern blot analysis, 1 μg of genomic DNA from each transformant was digested with SacI, resolved by gel electrophoresis, and transferred to nylon membranes. The egfp coding region was used as the template for the synthesis of random-primed [α-32P]dCTP-labeled probes. Southern hybridization and autoradiography were performed as described previously (4).
Genomic PCR.Genomic PCR was performed using 100 ng of genomic DNA from each transformant as the template. The forward primer corresponded to the mnp1 promoter sequence from position −1,412 to −1,390. The reverse primer corresponded to the egfp coding sequence 40 bp downstream of the start codon. PCR products were purified using a gel extraction kit (QIAGEN), and their sequences were determined.
RESULTS
Construction of expression plasmids.The restriction map of pUMiGM3′, which includes the egfp expression cassette consisting of the 1,528-bp mnp1 promoter, followed by the intron-containing egfp coding region and the 250-bp mnp1 3′ UTR, subcloned into pUB, is shown in Fig. 1 (31). The S. commune ura1 gene in pUB served as a selectable marker for transformation of the P. chrysosporium Ura11 auxotrophic strain (1). The various pUMiGM3′ derivatives containing deletions, an insertion, and a translocation in the mnp1 promoter region were sequenced to confirm the accuracy of the constructions.
GFP expression in transformants.Transformation of P. chrysosporium Ura11 protoplasts with 1 μg of pUMiGM3′ or a mutant derivative plasmid resulted in approximately 100 prototrophic transformants, of which 40 were isolated. GFP expression was screened by growing the transformants for 7 days at 37°C in HCLN stationary cultures supplemented with 180 μM MnSO4 and subsequently measuring the fluorescence intensity of the cell extract. Background fluorescence was determined in transformants obtained with the vector pUB. At least two transformation experiments, with characterization of the transformants, were performed with each plasmid. Significant expression of GFP was exhibited by approximately 25% of the isolated transformants obtained with each plasmid, with the exception of pUMiGM3′-A (see below). The levels of GFP fluorescence varied among individual transformants, and only transformants with fluorescence levels greater than three times the background were examined further. Five to eight transformants from each plasmid transformation that exhibited the highest fluorescent intensity were further purified by fruiting and isolating single basidiospore progeny (6, 19). GFP fluorescence was determined for the purified transformants grown in HCLN cultures with and without supplemented Mn2+. None of the transformants exhibited GFP expression when grown in HCHN cultures.
GFP expression with pUMiGM3′-A, pUMiGM3′-K, and pUMiGM3′-E.Large deletions from the 5′ end of the 1,528-bp mnp1 promoter in pUMiGM3′ resulted in the plasmids pUMiGM3′-A, pUMiGM3′-K, and pUMiGM3′-E, which contained 80, 521, and 774 bp of the mnp1 promoter, respectively (Fig. 2). Transformants (T) carrying pUMiGM3′-A exhibited only background GFP fluorescence under all culture conditions, suggesting that this 80-bp proximal mnp1 promoter fragment was unable to drive egfp expression. After 7 days of growth in HCLN medium containing Mn2+, transformants carrying pUMiGM3′-E or pUMiGM3′-K had lower levels of GFP fluorescence than transformants carrying pUMiGM3′. As shown in Fig. 2, when grown in HCLN medium supplemented with 180 μM Mn2+, the GFP fluorescence intensity from the two most fluorescent transformants carrying pUMiGM3′ (T-5 and T-11) was 2.5 to 4 times stronger than that from the two most fluorescent transformants carrying pUMiGM3′-K (T-11 and T-34) or pUMiGM3′-E (T-7 and T-31), suggesting that these deletions in the mnp1 promoter may have affected the overall expression of GFP.
Transformants carrying pUMiGM3′or pUMiGM3′-E exhibited significant GFP fluorescence only in HCLN cultures supplemented with Mn2+ (Fig. 2) (31). In contrast, transformants carrying pUMiGM3′-K exhibited GFP expression in HCLN cultures grown in the absence or in the presence of exogenous Mn2+. As shown in Fig. 2, transformants T-5 and T-11 (pUMiGM3′) and transformants T-7 and T-31 (pUMiGM3′-E) exhibited background levels of fluorescence when grown in HCLN medium without exogenous Mn2+, whereas the levels of GFP fluorescence from transformants T-11 and T-34 (pUMiGM3′-K) grown in HCLN cultures without Mn2+ were comparable to those in HCLN cultures supplemented with 180 μM Mn2+. This suggested that the mnp1 promoter sequence between the EagI and the KpnI sites might be responsible for Mn2+-dependent regulation of mnp1 promoter activity.
GFP expression with pUMiGM3′-ΔEK, pUMiGM3′ΔE197, pUMiGM3′ΔK(−48), and pUMiGM3′Δ48A.In HCLN cultures supplemented with 180 μM Mn2+, the levels of GFP fluorescence exhibited by transformants carrying pUMiGM3′-ΔEK, containing a 253-bp deletion of the mnp1 promoter between the EagI and the KpnI sites (Fig. 3A), were similar to those in transformants carrying pUMiGM3′. However, unlike transformants carrying pUMiGM3′, transformants carrying pUMiGM3′-ΔEK also exhibited significant levels of GFP fluorescence in HCLN cultures without exogenous Mn2+ (Fig. 3C). In transformants carrying pUMiGM3′, the ratio of GFP fluorescence intensity in cultures supplemented with 180 μM Mn2+ versus cultures without Mn2+ [GFP(+Mn)/GFP(−Mn)] was over 40. In contrast, in transformants carrying pUMiGM3′-ΔEK, this ratio ranged from 1.6 to 2.5 (Table 1). The fluorescence intensities of the three most fluorescent transformants carrying pUMiGM3′ (T-3, T-5, and T-11) and pUMiGM3′-ΔEK (T-3, T-6, and T-12) in 7-day-old HCLN cultures, with and without supplemented Mn2+, are shown in Fig. 3C. The fluorescence intensities of T-3, T-6, and T-12 (pUMiGM3′-ΔEK) in the absence of Mn2+ were approximately 50% of those in the presence of Mn2+. These data again suggest that the fragment between the EagI and KpnI sites in the mnp1 promoter contains sequences that are involved in Mn2+-dependent regulation of mnp1 promoter activity.
GFP(+Mn)/GFP(−Mn) ratios in transformants carrying pUMiGM3′ and its mutant derivativesa
To analyze the EagI-KpnI region in more detail, additional deletions were constructed. The plasmid pUMiGM3′ΔE197 contained a 197-bp deletion immediately downstream of the EagI site in the mnp1 promoter (Fig. 3A). The GFP expression profiles in transformants carrying pUMiGM3′ΔE197 were similar to those in transformants carrying pUMiGM3′, with a GFP(+Mn)/GFP(−Mn) ratio greater than 24 (Table 1). As shown in Fig. 3C, the three most fluorescent transformants carrying either pUMiGM3′ (T-3, T-5, and T-11) or pUMiGM3′ΔE197 (T-12, T-19, and T-31) exhibited similar levels of GFP fluorescence in HCLN cultures supplemented with 180 μM Mn2+ but only background levels of GFP fluorescence in HCLN cultures grown in the absence of Mn2+. Additional deletions of 150- and 100-bp fragments immediately downstream of the EagI site in the mnp1 promoter were also constructed. Transformants carrying these plasmids showed GFP expression profiles similar to those carrying pUMiGM3′ or pUMiGM3′ΔE197 (data not shown).
The plasmid pUMiGM3′ΔK(−48) contained a 48-bp deletion immediately upstream of the KpnI site in the mnp1 promoter (Fig. 3A). Thus, the deletions in pUMiGM3′ΔE197 and pUMiGM3′ΔK(−48) together encompassed the entire EagI-KpnI region. Transformants carrying pUMiGM3′ΔK(−48) exhibited GFP expression profiles that were similar to those in transformants carrying pUMiGM3′-ΔEK. The GFP(+Mn)/GFP(−Mn) ratios ranged from 1.5 to 2.7 in transformants carrying pUMiGM3′ΔK(−48) (Table 1).
The plasmid pUMiGM3′Δ48A contained a 48-bp sequence matching the 5′ end of mnp1 exon 6 in place of the 48-bp fragment that was deleted in pUMiGM3′ΔK(−48) (Fig. 3A and B). Transformants carrying pUMiGM3′Δ48A exhibited GFP expression profiles that were similar to those of transformants carrying pUMiGM3′-ΔEK or pUMiGM3′ΔK(−48). The GFP(+Mn)/GFP(−Mn) ratios ranged from 1.5 to 3.0 in transformants carrying pUMiGM3′Δ48A (Table 1). As shown in Fig. 3C, when grown in HCLN cultures without exogenous Mn2+, the three most fluorescent transformants carrying pUMiGM3′ΔK(−48) (T-12, T-19, and T-29) and the three carrying pUMiGM3′Δ48A (T-2, T-17, and T-36) exhibited approximately 40 to 60% of the GFP fluorescence intensity observed in HCLN cultures supplemented with 180 μM Mn2+. These data suggest that the 48-bp sequence immediately upstream of the KpnI site in the mnp1 promoter is responsible for Mn2+-regulated gene expression.
Northern blot analysis.Total RNA was extracted from transformants carrying either pUMiGM3′, pUMiGM3′-ΔEK, pUMiGM3′ΔE197, pUMiGM3′ΔK(−48), or pUMiGM3′Δ48A grown in 5-day-old HCLN cultures supplemented with or without 180 μM Mn2+. The RNA was resolved on a denaturing agarose gel, transferred to a nylon film, and probed with a 32P-labeled egfp coding region fragment, with the mnp1 cDNA, and with the gpd gene. As shown in Fig. 4A, significant amounts of egfp transcript were observed in transformants carrying either pUMiGM3′-ΔEK, pUMiGM3′ΔK(−48), or pUMiGM3′Δ48A, grown both in the presence and in the absence of exogenous Mn2+. In contrast, in transformants carrying pUMiGM3′ or pUMiGM3′ΔE197, egfp transcript was observed only in cells from HCLN cultures supplemented with 180 μM Mn2+. In all the transformants, egfp transcription levels correlated with GFP fluorescence levels (Fig. 3C and 4A).
Northern blot analysis of egfp, mnp1, and gpd transcript levels in transformants carrying pUB, pUMiGM3′, pUMiGM3′-ΔEK, pUMiGM3′ΔE197, pUMiGM3′ΔK(−48), and pUMiGM3′Δ48A. Total RNA was extracted from 5-day-old HCLN cultures grown either in the absence of exogenous Mn2+ (−Mn) or supplemented with 180 μM Mn2+ (+Mn). The RNA blot was probed with the following 32P-labeled genes: egfp coding region (A), mnp1 cDNA (B), or gpd (C), as described in the text.
As shown in Fig. 4B, probing of the Northern blot with 32P-labeled mnp1 cDNA revealed significant amounts of mnp1 transcript in all transformants, but only in HCLN cultures supplemented with 180 μM Mn2+. This indicates that the transformation of P. chrysosporium with the various egfp expression plasmids did not affect the activation of endogenous mnp1 gene transcription by Mn2+. The loading control shown in Fig. 4C demonstrates the presence of gpd transcripts in all transformants grown with or without Mn2+.
These data indicate that deletion of the 253-bp EagI-KpnI region, deletion of the 48-bp fragment upstream of the KpnI site, or replacement of the 48-bp fragment with an unrelated sequence, all affect the Mn2+-regulated transcription of egfp directed by the mnp1 promoter, and the data confirm that the 48-bp sequence immediately upstream of the KpnI site is responsible for Mn2+-dependent regulation.
Southern blot analysis.Southern blot analysis was performed on genomic DNA from each of the transformants shown in Fig. 3 and 4. In all these transformants, the egfp-expressing plasmids were integrated into the chromosomes. No autonomously replicating plasmids were detected. In addition, the copy numbers of the integrated plasmids appeared similar in all of these transformants (data not shown). Genomic PCR was performed on each transformant shown in Fig. 3 and 4, amplifying a mnp1 promoter fragment from 1.4 kb upstream of the translation start codon to 40 bp downstream of the egfp start codon. Sequencing of the PCR products confirmed the accuracy of the mutations in the integrated plasmids (data not shown).
GFP expression in pUMiGM3′-48N.The 48-bp DNA fragment immediately upstream of the KpnI site in the mnp1 promoter was deleted and reinserted into a unique NsiI site residing 120 bp downstream of the KpnI site, to yield pUMiGM3′-48N (Fig. 5). Transformants carrying pUMiGM3′-48N had significant GFP fluorescence only in HCLN cultures supplemented with 180 μM Mn2+. The GFP(+Mn)/GFP(−Mn) ratio ranged from 12 to 34 in transformants carrying pUMiGM3′-48N (Table 1). As shown in Fig. 5, the three most fluorescent transformants carrying pUMiGM3′-48N (T-13, T-23, and T-34) and the three carrying pUMiGM3′ (T-3, T-5, and T-11) expressed GFP only in HCLN cultures containing Mn2+, whereas transformants carrying pUMiGM3′ΔK(−48) expressed GFP in HCLN cultures both in the presence and in the absence of Mn2+. Genomic PCR was performed on transformants carrying pUMiGM3′-48N, confirming the new location of the 48-bp fragment in the mnp1 promoter (data not shown). These results suggest that the 48-bp sequence itself, rather than its specific location, is responsible for Mn2+-dependent regulation of mnp1 promoter activity.
GFP expression in transformants carrying pUMiGM3′ΔK(−48) and pUMiGM3′-48N. The upper panel shows the modified mnp1 promoter regions in pUMiGM3′ΔK(−48) and pUMiGM3′-48N. Dashed line, deleted sequence; triangle, insert location. The lower panel shows the fluorescence intensities of the three most fluorescent transformants carrying each construct, grown in HCLN cultures with or without added Mn2+. Data were calculated as described in the legend to Fig. 2.
GFP expression in pUMiGM3′Δ24 and pUMiGM3′Δ33.The two plasmids, pUMiGM3′Δ24 and pUMiGM3′Δ33, contained internal deletions in the 48-bp DNA fragment immediately upstream of the KpnI site in the mnp1 promoter. pUMiGM3′Δ24 contained a 24-bp deletion located between 48 and 25 bp upstream of the KpnI site, whereas pUMiGM3′Δ33 contained a 33-bp deletion immediately upstream of the KpnI site (Fig. 6). The levels of GFP fluorescence exhibited by the three most fluorescent transformants carrying pUMiGM3′Δ24 (T-6, T-10, and T-29) and the three most fluorescent transformants carrying pUMiGM3′Δ33 (T-8, T-18, and T-36) are shown in Fig. 6. In HCLN cultures supplemented with 180 μM Mn2+, transformants carrying either plasmid exhibited significant GFP fluorescence. However, in HCLN cultures grown without exogenous Mn2+, significant GFP fluorescence was only observed in transformants carrying pUMiGM3′Δ33. The GFP(+Mn)/GFP(-Mn) ratio ranged from 1.6 to 5 in transformants carrying pUMiGM3′Δ33, whereas the ratio was more than 15 in transformants carrying pUMiGM3′Δ24 (Table 1). These results suggest that the 33-bp fragment immediately upstream of the KpnI site plays an important role in Mn2+-dependent regulation of mnp1 promoter activity.
GFP expression in transformants carrying pUMiGM3′Δ24 and pUMiGM3′Δ33. The upper panel shows the sequences of the 48-bp fragment (capital letters) immediately upstream of the KpnI site in the mnp1 promoter and of its mutant derivatives in pUMiGM3′Δ24 and pUMiGM3′Δ33. The stars represent the deleted nucleotides. The putative AP-2 binding site is shown in bold. The nucleotides that were mutated are underlined. The lower panel shows the fluorescence intensities of the three most fluorescent transformants carrying each construct, grown in HCLN cultures with or without added Mn2+. Data were calculated as described in the legend to Fig. 2.
DISCUSSION
MnP is a major extracellular component of the lignin and pollutant degradation system produced by the white-rot fungus P. chrysosporium (17, 19, 37, 38). The transcription of mnp genes in this fungus is activated by nitrogen depletion and Mn2+ supplementation (7, 8, 12, 13, 16, 39). In this study we have introduced deletion, replacement, and translocation mutations into the P. chrysosporium mnp1 promoter and examined the effects of these mutations on mnp1 promoter-directed egfp expression under Mn2+-sufficient and Mn2+-deficient culture conditions.
Previously we reported that a 1,528-bp mnp1 promoter fragment was sufficient for driving egfp expression under Mn2+-sufficient and nitrogen-limiting conditions identical to those required for endogenous MnP production (31). Here we report that deletion of a 253-bp EagI-KpnI fragment, residing 521 bp upstream of the translation start codon, results in accumulation of egfp transcript and significant GFP production under both Mn2+-deficient and Mn2+-sufficient conditions (Fig. 3 and 4). This same phenotype is observed upon deletion of a 48-bp fragment immediately upstream of the KpnI site, but not from deletion of the remaining 197-bp fragment within the EagI-KpnI region, suggesting that the 48-bp fragment contains a cis-acting sequence(s) which is responsible for Mn2+-dependent regulation of mnp1 promoter activity (Fig. 3 and 4). Since the sequence deletions could cause an alteration in DNA configuration that might affect mnp1 promoter activity, a replacement mutation for the 48-bp fragment was constructed which maintained the length of the mnp1 promoter. This replacement mutation exhibited a Mn2+-independent GFP expression profile, similar to that observed with deletions of either the entire EagI-KpnI fragment or the 48-bp fragment. In addition, the entire 48-bp fragment was moved 120 bp downstream from its original location. The mnp1 promoter containing this translocation mutation efficiently drives egfp expression under Mn2+-sufficient conditions, but not under Mn2+-deficient conditions, similar to the wild-type mnp1 promoter. These data suggest that the nucleotide sequence of the 48-bp fragment is responsible for Mn2+-dependent regulation of mnp1 promoter activity.
Since deletion or replacement of the 48-bp fragment results in derepression of egfp expression under Mn2+-deficient conditions, we propose that, in the absence of Mn2+, negative control is exerted at a site within the 48-bp fragment and that this control is released upon Mn2+ supplementation. Negative control mechanisms have been observed in the regulation of genes involved in siderophore biosynthesis and siderophore-mediated iron uptake in response to iron status in Aspergillus nidulans (21), Neurospora crassa (49), Ustilago maydis (47), and Penicillium chrysogenum (21).
Deletion of a 33-bp fragment at the 3′ end of the 48-bp sequence results in significant GFP expression under Mn2+-deficient conditions, whereas deletion of a 24-bp fragment at the 5′ end of the 48-bp sequence does not. This suggests that the 33-bp DNA sequence immediately upstream of the KpnI site in the mnp1 promoter contains the Mn2+-responsive element(s). To our knowledge this is the first promoter sequence containing an Mn2+-responsive element(s) to be characterized in any eukaryotic organism.
Under Mn2+-deficient conditions, the GFP fluorescence intensities exhibited by transformants containing the deletion of the entire EagI-KpnI fragment, the deletion of the 48-bp fragment, or the 48-bp replacement fragment were approximately 50 to 60% of those observed under Mn2+-sufficient conditions (Fig. 3; Table 1). This suggests that there might be another Mn2+-dependent regulatory element(s) in the mnp1 promoter residing outside of the 48-bp segment that we identified.
In our P. chrysosporium transformation experiments, the plasmids integrate into the fungal chromosomes at predominantly ectopic sites (1, 2, 4, 18). The integration sites may affect the expression of genes carried by the plasmids, which may explain why only 25% of the transformants carrying egfp expression plasmids exhibited significant GFP fluorescence. Among the transformants with significant GFP fluorescence, the intensity varied under both Mn2+-sufficient and Mn2+-deficient conditions. However, the ratios of GFP fluorescence intensities in 7-day-old HCLN cultures with or without 180 μM Mn2+, GFP(+Mn)/GFP(−Mn), were consistently either high or low among transformants carrying the same plasmid (Table 1). The GFP (+Mn)/GFP(−Mn) ratios among transformants carrying pUMiGM3′-ΔEK, pUMiGM3′ΔK(−48), pUMiGM3′Δ48A, or pUMiGM3′Δ33 were low (between 1.5 and 5), and variations in the values were relatively small. In contrast, the GFP(+Mn)/GFP(−Mn) ratios among transformants carrying pUMiGM3′, pUMiGM3′ΔE197, pUMiGM3′-48N, or pUMiGM3′Δ24 were generally high (larger than 12). In several transformants carrying pUMiGM3′, pUMiGM3′ΔE197, or pUMiGM3′Δ24, the fluorescence intensities exhibited under Mn2+-deficient conditions were less than or equal to the background fluorescence intensities exhibited in transformants carrying the control vector, pUB. Therefore, in these cases, the GFP(+Mn)/GFP(−Mn) ratios have not been calculated. Most importantly, the differences in the GFP(+Mn)/GFP(−Mn) ratios calculated for transformants carrying pUMiGM3′, pUMiGM3′ΔE197, pUMiGM3′-48N, or pUMiGM3′Δ24 and the ratios calculated for transformants carrying pUMiGM3′-ΔEK, pUMiGM3′ΔK(−48), pUMiGM3′Δ48A, or pUMiGM3′Δ33 were significant and clearly result from the mutations constructed in the mnp1 promoter region of individual egfp expression plasmids.
Comparisons with the intact mnp1 promoter showed that similar levels of GFP expression were observed under Mn2+-sufficient, nitrogen-limited conditions with mnp1 promoters containing mutations within the EagI-KpnI region (Fig. 3, 5, and 6). However, deleting the mnp1 promoter sequence upstream of the EagI site resulted in lower levels of GFP expression (Fig. 2). In addition, deletion of the mnp1 promoter to a position 80 bp upstream of the start codon resulted in no GFP production (Fig. 2). These data suggest that promoter elements residing outside of the EagI-KpnI region contribute to the overall efficiency of mnp1 promoter activity. Furthermore, no transformant characterized in this study showed significant GFP fluorescence in nitrogen-sufficient cultures, suggesting that the nitrogen status-dependent regulation of mnp1 promoter activity requires elements other than the 33-bp fragment responsible for the Mn2+-dependent regulation.
Previous sequence analysis has shown that both the P. chrysosporium mnp1 and mnp2 genes (17, 18, 32) contain short promoter sequences matching the consensus sequence of MREs, as defined in mammalian metallothionein genes (9, 11). Metallothioneins are small, cysteine-rich proteins that bind and store transition metal ions such as Cd2+, Zn2+, and Cu+ in mammalian cells (34, 45). Six MRE-like elements are found in the sequenced mnp1 promoter. Four of these, located near the translation start codon, are arranged into two pairs with 4-bp palindromes (18, 20). These two pairs are designated MREd (with the sequence GCGTGCACGC) and MREp (with the sequence GTGTGCACGC), residing at 315 and 91 bp upstream of the start codon, respectively (Fig. 1). Point mutations that change the center four bases, TGCA to TTAA, have been constructed in either MREd or MREp and in both MRE pairs within the mnp1 promoter in pUMiGM3′. Insertion mutations also have been constructed by inserting 10-bp linkers (GCTGATCACG) into the center of either MREp or MREd and into both MRE pairs. Transformants carrying these egfp expression plasmids exhibit regulation of GFP expression by Mn2+ supplementation and nitrogen limitation, similar to transformants carrying pUMiGM3′ (unpublished data). These results suggest that the putative MREs are not responsible for Mn2+-dependent regulation of mnp1 promoter activity. Recently it was reported that in the basidiomycete Trametes versicolor the addition of 200 μM Mn2+ to Mn2+-deficient cultures resulted in an 8-fold and a 250-fold increase in mnp1 and mnp2 transcripts, respectively (24). In this fungus, putative MREs are found only in the mnp1 promoter, suggesting that Mn2+-dependent regulation of mnp gene expression in T. versicolor also does not require the putative MRE sequences.
The 33-bp P. chrysosporium mnp1 promoter sequence, containing a Mn2+-responsive cis element(s) which we identified, possesses a high G+C content (67%). Moreover, there are two identical repeating sequences (GCGTTGGG) in this fragment, conforming to a binding site for AP-2 (11, 17, 18) (Fig. 3 and 6). In higher eukaryotes, AP-2 is a family of cell-type-specific and developmentally regulated transcription factors which are critical regulators of gene expression during development and carcinogenesis (25, 33). In mammals, AP-2 apparently mediates the activation of metabolic processes by two different signal transduction pathways, one using protein kinase C and the other using cyclic AMP (23). However, evidence of AP-2 involvement in Mn2+-dependent gene expression regulation is lacking. An earlier assertion hypothesized that AP-2 may be important for the regulation of LiP- and MnP-encoding gene expression in white-rot fungi by nitrogen limitation (10). However, our results have shown that the deletion of the 33-bp mnp1 promoter sequence does not affect the regulation of mnp1 promoter activity by nitrogen status, suggesting that the two AP-2 sites found within this fragment are not involved in the nitrogen-dependent regulatory response. Furthermore, in pUMiGM3′Δ24, three nucleotides of the first putative AP-2 binding site within the 33-bp mnp1 promoter sequence were mutated (Fig. 6). Transformants carrying pUMiGM3′Δ24 exhibited Mn2+-dependent GFP expression as shown in transformants carrying pUMiGM3′, suggesting that, if the putative AP-2 binding sites were important for Mn2+-dependent regulation of mnp1 promoter activity, perhaps only one intact site would be required.
Sequences that show strong homology to the 33-bp mnp1 promoter sequence can be found in the P. chrysosporium mnp2 (32) (GenBank accession number S69963 ) and mnp3 (3) (GenBank accession number U70998 ) promoter regions, and these sequences are shown in Fig. 7. The strong homology among these three sequences suggests that the putative Mn2+-responsive element may be conserved among P. chrysosporium mnp genes. Future experiments will focus on defining those nucleotides in the 33-bp fragment that are essential for the regulation of mnp1 promoter activity in response to Mn2+ supplementation and also on characterization of a trans-acting transcription factor(s) that interacts with this sequence element.
Nucleotide sequences in the P. chrysosporium mnp2 (B) and mnp3 (C) promoter regions that show high homology with the 33-bp sequence (A) from the mnp1 promoter involved in Mn2+-dependent gene expression regulation. The 33-bp sequence was aligned individually with P. chrysosporium mnp2 promoter sequence (GenBank accession number S69963 ; 1,287 bp in length) and mnp3 promoter sequence (GenBank accession number U70998 ; 1,192 bp in length) with the ClustalW alignment program in Macvector 6.5 software (Oxford Molecular Group). The homologous regions were subjected to a multiple alignment with the 33-bp mnp1 promoter sequence using the same program. The shaded areas represent the homologous motifs. Letters in bold indicate the nucleotides that are conserved in two or more sequences. The consensus sequence is shown at the bottom of the aligned sequences.
ACKNOWLEDGMENTS
This research was supported by grant MCB-9723725 from the National Science Foundation and grant DE-FG-03-96ER20235 from the Division of Energy Biosciences, U.S. Department of Energy, to M.H.G.
FOOTNOTES
- Received 8 December 2003.
- Accepted 31 March 2004.
- Copyright © 2004 American Society for Microbiology
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