This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.
Agricola
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2008, p. 6006-6016, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01188-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Asexual Cephalosporin C Producer Acremonium chrysogenum Carries a Functional Mating Type Locus{triangledown}

Stefanie Pöggeler,1,{ddagger} Birgit Hoff,2,3,{ddagger} and Ulrich Kück2,3*

Abteilung Genetik Eukaryotischer Mikroorganismen, Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany,1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany,2 Christian Doppler-Labor, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany3

Received 28 May 2008/ Accepted 30 July 2008


arrow
ABSTRACT
 
Acremonium chrysogenum, the fungal producer of the pharmaceutically relevant β-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type (MAT) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the {alpha}-box domain gene, MAT1-1-1 and MAT1-1-2, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (AcMAT1-1) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating type gene, AcMAT1-1-3, encoding a high-mobility-group domain protein. The {alpha}-box domain sequence of AcMAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the AcMAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the Pa{Delta}MAT strain). After fertilization with a P. anserina MAT1-2 (MAT+) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum.


arrow
INTRODUCTION
 
Acremonium chrysogenum (formerly named Cephalosporium acremonium) (22) is the industrial producer of the pharmaceutically relevant β-lactam antibiotic cephalosporin C (64). A. chrysogenum was isolated from seawater close to a sewage outfall area at Cagliari (Sardinia, Italy) in 1945 by Giuseppe Brotzu (6) and was found to produce, among other antibiotics, a β-lactam compound designated cephalosporin C, which is structurally related to penicillin (1). Today, cephalosporin derivatives are widely used in the treatment of infectious diseases and are one of the world's major biotechnological products, with a total world market of about $10 billion (5). The biosynthetic pathway for cephalosporin C is well characterized both genetically and biochemically and involves eight enzymatic steps that are catalyzed by seven different enzymes (38, 64). In addition, the expression of the corresponding biosynthesis genes is known to be controlled by several different transcriptional regulators (16, 63-66).

The genus Acremonium is a highly polyphyletic taxon containing distantly related fungi, including species associated with at least three or four ascomycete orders. Phylogenetic analyses revealed previously that A. chrysogenum shows affiliation with the order Hypocreales (25). To date, A. chrysogenum propagation has been accepted to be strictly asexual, by the formation of conidiospores and arthrospores (27, 42), and no associated teleomorph showing sexual propagation has been found. Thus, strains for improved industrial cephalosporin production can be obtained only by conventional mutagenesis and selection procedures or by molecular genetic techniques (29, 60, 72). However, the ability to mate fungi under controlled laboratory conditions would be a valuable tool for genetic analysis, as well as for conventional strain improvement programs (52).

In fungi, sexual reproduction is regulated by a genomic region referred to as the mating type (MAT) locus (20). In self-sterile (heterothallic) species, mating occurs between morphologically identical partners that are distinguished only by their MAT loci. The MAT locus consists of two dissimilar DNA sequences in the mating partners, termed the MAT1-1 and MAT1-2 idiomorphs (71). The MAT1-1 locus invariably contains the MAT1-1-1 gene, encoding a protein with an {alpha}-box domain. In most of the sordariomycetes, two other genes are also located in the MAT1-1 locus: (i) the MAT1-1-2 gene, encoding a protein with an HPG domain haboring the three invariant residues histidine, proline, and glycine, and (ii) the MAT1-1-3 gene, encoding a protein with a high-mobility-group (HMG) domain as a DNA-binding motif. The hallmark of the MAT1-2 locus is the MAT1-2-1 gene, encoding a protein with an HMG domain. In addition to the MAT1-2-1 gene, other genes may also be present at the MAT1-2 locus (14, 15, 70). In contrast to the genomes of heterothallic species, the genomes of self-fertile (homothallic) filamentous ascomycetes contain genes indicative of both mating types that can be either linked or unlinked (21, 55, 61, 80).

Moreover, several asexual filamentous fungi have been reported previously to contain functional, constitutively transcribed MAT genes. Among these are plant and human pathogens like Bipolaris sacchari and Aspergillus fumigatus, as well as biotechnologically relevant fungi such as Penicillium chrysogenum and Aspergillus oryzae (3, 17, 21, 26, 32, 49, 50, 67, 76). Analyses of the genome sequences of the asexual human pathogens Aspergillus fumigatus and Penicillium marneffei and that of Penicillium chrysogenum revealed the presence of transcriptionally active genes associated with sexual reproduction, including genes for pheromone production and recognition (21, 26, 49, 50, 76). These reports indicate that the absence of sexual reproduction is not due to disruptive mutations within MAT genes or other sex-related genes.

Here, we provide the first direct evidence of an A. chrysogenum MAT1-1 (AcMAT1-1) locus with at least three transcriptionally expressed genes. Furthermore, we provide a functional analysis of this AcMAT1-1 locus, which induced the formation of fruiting bodies and ascospores in the heterothallic ascomycete Podospora anserina. Moreover, we identified the homologue of a pheromone receptor gene that is known to function in mating and signaling in sexually reproducing filamentous ascomycetes, as well as homologues of a putative pheromone response pathway and putative pheromone-processing enzymes. To the best of our knowledge, this is the first report of an industrially relevant filamentous ascomycete for which the functionality of MAT genes is demonstrated by expression in a heterologous host. Our findings therefore open up the intriguing possibility of inducing mating and sexual reproduction as alternative strain improvement strategies for A. chrysogenum.


arrow
MATERIALS AND METHODS
 
Strains and culture conditions.
Escherichia coli strain K-12 XL1-Blue was used for general plasmid construction and maintenance (7). A list of all A. chrysogenum isolates used, with source details, is provided in Table 1. All strains were routinely maintained at 27°C on solid complex culture medium as described previously (41). P. anserina strains expressing MAT1-1 (MAT–) and MAT1-2 (MAT+) were derived from homokaryotic spores of P. anserina wild-type strains (18) containing only one of the two mating types. The P. anserina MAT (PaMAT) deletion mutant used in this study, Pa{Delta}MAT, was generated by replacing the PaMAT1-2 idiomorph by the ura5+ transformation marker (11). Crosses and maintenance of P. anserina strains were performed on standard cornmeal agar (BMM) at 27°C under constant light.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Characteristics of A. chrysogenum strains used in this study, including mating types, loci of isolation, and sources

Isolation of the AcMAT1-1 locus.
The expressed sequence tag (EST) library from A. chrysogenum (Sandoz GmbH, Kundl, Austria) was used to identify an A. chrysogenum homologue of the MAT1-1-1 gene critical for mating in other ascomycetes. To clone the entire AcMAT1-1 locus, the sequences of EST clone AcEST.4.5311 containing the putative MAT1-1-1 gene were used to design primer pair MAT1-f (5'-CGGCCTCTGAACGGCTTCATG-3') and MAT1-r (5'-GTCGTCGTTCTCGACATTCCA-3'). These primers were used in a rapid screening procedure (54) to screen an indexed A. chrysogenum cosmid library (16). The screening resulted in the isolation of cosmid P2-G5, which was subsequently used for the subcloning of the entire AcMAT1-1 locus by standard protocols (62). The subcloning of hybridizing DNA fragments from cosmid clone P2-G5 led to the construction of derivatives of the plasmid pDrive (Qiagen, Germany). Both strands of plasmid inserts were sequenced with universal primers T7 and SP6 (MWG-Biotech, Germany). The completion of the sequence was achieved by the synthesis of internal sequencing primers corresponding to regions where coverage was insufficient. Finally, the obtained partial sequences were combined into a single consensus sequence covering, in total, 12,076 bp of the AcMAT1-1 locus and adjacent regions.

Generation of cosmid P2-G5-hph for transformation of P. anserina.
The screening of an A. chrysogenum cosmid library led to the isolation of cosmid P2-G5 carrying the complete AcMAT1-1 locus. In order to use this cosmid for the direct transformation of the Pa{Delta}MAT strain, we randomly integrated the hph resistance gene under the control of the trpC promoter of Aspergillus nidulans by using an in vitro recombination approach as described previously (16). The recombinant cosmid P2-G5-hph was examined by PCR and Southern analyses for the integrity of the AcMAT1-1 locus.

Transformation of P. anserina.
For transformation procedures, the Pa{Delta}MAT strain was cultivated in static liquid complete medium (45) at 27°C under constant light. The preparation and transformation of P. anserina protoplasts were performed as described previously (47). Fungal protoplasts were transformed with 15 µg of cosmid DNA, and the selection of transformants occurred on solid BMM medium supplemented with 50 U of hygromycin B/ml. The segregation of antibiotic resistance phenotypes in the sexual crosses was scored on the same medium.

Preparation of nucleic acids and DNA hybridization.
For DNA and RNA extraction, A. chrysogenum strains were cultivated in liquid complex culture medium at 27°C with (180-rpm) or without shaking and P. anserina strains were grown in static liquid complete medium (45). After growth, resulting mycelia were removed, flash frozen, and ground under liquid nitrogen prior to nucleic acid extraction. Fungal genomic DNA and RNA were extracted by phenol-chloroform and chloroform-isoamyl alcohol methods as described previously (46). Southern blotting was performed with GeneScreen hybridization transfer membrane according to the instructions of the manufacturer (PerkinElmer, Boston, MA). Filters were hybridized with [{alpha}-32P]dCTP-labeled probes by using standard methods (62).

PCR and semiquantitative RT-PCR for expression analyses.
For use in semiquantitative reverse transcription (RT)-PCR, cDNA was synthesized as described previously (46), with the following modifications: 2-µg aliquots of total RNA were treated with 3.5 U of DNase I according to the recommendations of the manufacturer (Invitrogen, Germany), and RT was performed with 400 U of SuperScript II (Invitrogen, Germany) and deoxynucleoside triphosphates at a concentration of 0.33 mM. As a control for successful DNase treatment, each RT step was carried out twice, once with and once without reverse transcriptase. All samples were used together with genomic DNA as templates for the expression analysis of the PaMAT and AcMAT genes with the following primers: (i) for the AcMAT1-1-1 gene, primers MAT1-f and MAT1-r; (ii) for the AcMAT1-1-2 gene, primers MAT2-f (5'-CCGAGTCGCTCAAGAATGGCAA-3') and MAT2-r (5'-TCATGGAAGACGCAAGCATA-3'); (iii) for the AcMAT1-1-3 gene, primers MAT3-f (5'-GTGCCTGAGACATTCGAATGG-3') and MAT3-r (5'-CGATCGTACCAGAACTGTCGCT-3'); (iv) for the PaMAT1-1-1 (FMR1) gene, primers FMR1-f (5'-CCAAGAAGAAGGTCAACGGTT-3') and FMR1-r (5'-GGAACTTGCGTGTTGATACAGAT-3'); and (v) for the PaMAT1-2-1 (FPR1) gene, primers FPR1-f (5'-CCCTAACGCCTACATTCTTTA-3') and FPR1-r (5'-GCACAGTCGACTCCATTGTTT-3').

PCR and RT-PCR analyses were conducted using a total volume of 50 µl containing 150 µg of genomic DNA or 200 ng of cDNA, 10 pmol of each primer, 1 mM (each) deoxynucleoside triphosphates, and 5 U of HotMaster Taq polymerase (5 Prime, Germany). PCR conditions were as follows: 2 min at 94°C; 40 cycles of 1 min at 94°C, 1 min at 60 to 65°C, and 5 min at 68°C; and a final step of 15 min at 25°C.

Microscopy.
For microscopic studies, P. anserina transformants were cultivated on solid BMM medium at 27°C under constant light. Morphologies of perithecia from P. anserina wild-type and transformant strains were analyzed using an Axiophot microscope (Carl Zeiss, Jena, Germany). Images were captured with an Axiovision digital system including the Zeiss Axiophot camera. Recorded pictures were processed with Adobe Photoshop CS2 software.

Sequence and phylogenetic analyses.
The EST library from A. chrysogenum was used to identify A. chrysogenum homologues of genes critical for mating and pheromone signaling in other ascomycetes. Sequences of the cDNAs were obtained by custom sequencing carried out by Sandoz GmbH (Kundl, Austria). Fungal protein sequences from different sources were used as query sequences in searches with the basic local alignment search tool BLAST (2). For homology searching, the TBLASTN program was used. The E value cutoff used to select homologues was 1e–6. For validation of the identified A. chrysogenum genes, a bidirectional best-hit analysis was performed by using the DNA sequence of the identified A. chrysogenum EST clone as a query in a BLASTX search of the SwissProt/TrEMBL/TrEMBL_NEW database at the Swiss Institute of Bioinformatics (http://www.ch.embnet.org).

Protein sequence data were obtained from the NCBI Entrez public databases (http://www.ncbi.nlm.nih.gov/entrez/), and sequences were aligned using the ClustalX program (68). Phylogenetic analysis was carried out with programs contained in the program package PHYLIP version 3.6 (19). The same alignment was used for phylogenetic analysis performed by the distance matrix method and a maximum-parsimony method. Distance matrices were calculated using the program PROTDIST and were then used for constructing trees with the neighbor-joining program NEIGHBOR. The program PROTPARS was used for constructing maximum-parsimony trees. Statistical significance was evaluated by bootstrap analysis with 1,000 iterations of bootstrap samplings generated with SEQBOOT. A majority-rule consensus tree was subsequently generated using the program CONSENSE. The consensus trees were graphically displayed using TreeView (48).


arrow
RESULTS
 
Identification of EST clones coding for AcMAT proteins.
To determine whether MAT genes are present in A. chrysogenum, we performed a BLAST analysis of the sequences present in an EST library from A. chrysogenum strain ATCC 14553. A TBLASTN search using the amino acid sequence corresponding to the Neurospora crassa MAT1-1-1 (MATA-1) {alpha}-box domain gene as the query sequence revealed the presence of an 827-bp cDNA clone (AcEST.4.5311) encoding a polypeptide with 49% amino acid similarity to the Neurospora crassa MAT1-1-1 protein. A BLASTX search of the EMBL databases with the cDNA sequence of clone AcEST.4.5311 as the query showed similarity between the sequence of the putative A. chrysogenum {alpha}-box protein and those of {alpha}-box mating type proteins of other filamentous ascomycetes. The highest degrees of sequence similarity (with E values of 6e–41 and 3e–37) were those to MAT1-1-1 of the entomopathogenic fungus Cordyceps takaomontana (anamorphs, Paecilomyces tenuipes and Isaria tenuipes) (77-79) and MAT1-1-1 of the asexual fungal pathogen Fusarium poae (32). By using the sequence of the MAT1-1-2 protein of Cordyceps takaomontana as the query sequence for a TBLASTN search, the product of a 549-bp EST clone (AcEST.4.20371) was found to show the highest degree of sequence identity. A BLASTX search of the EMBL databases with the nucleotide sequence of clone AcEST.4.20371 revealed a high degree of similarity to the sequence encoding the MAT1-1-2 protein of Claviceps purpurea (78).

A TBLASTN search with MAT1-2-1 proteins from different filamentous ascomycetes failed to identify an EST clone encoding an HMG domain with significant similarity (data not shown). Thus, we concluded that A. chrysogenum ATCC 14553 is a MAT1-1 strain.

Cloning of the entire MAT1-1 locus of A. chrysogenum ATCC 14553.
To clone the entire AcMAT1-1 locus, the sequences of EST clone AcEST.4.5311 containing the putative AcMAT1-1-1 gene were used to design the primer pair MAT1-f/MAT1-r. Subsequent genomic PCR conducted with this primer pair produced a 336-bp amplicon, and DNA sequencing confirmed its identity (Fig. 1). The primers were then used in a rapid screening procedure (54) to screen an indexed A. chrysogenum cosmid library. The isolated clone P2-G5 was subsequently used for the subcloning of the entire AcMAT1-1 locus. The sequence of the subcloned DNA, covering in total 12,076 bp of the mating type locus and adjacent regions, was determined. Combined computer and RT-PCR analyses of this sequence resulted in the identification of three putative MAT genes which, according to the nomenclature of Turgeon and Yoder (71), were termed AcMAT1-1-1, AcMAT1-1-2, and AcMAT1-1-3 (Fig. 1).


Figure 1
View larger version (4K):
[in this window]
[in a new window]

  		FIG. 1. Schematic illustration of the AcMAT1-1 locus and its flanking regions. The positioning and transcriptional directions of the mating type genes (AcMAT1-1-1, AcMAT1-1-2, and AcMAT1-1-3) and the flanking genes (AcAPN2 and AcSLA2), as well as AcEST.4.744, are indicated by arrows. Introns are shown in black. E, EcoRI; B, BamHI; H, HindIII.

The AcMAT1-1-1 open reading frame (ORF) encodes a polypeptide of 375 amino acids (aa) and contains a single intron of 55 bp, which habors typical A. chrysogenum splice sites (30). Comparison of the predicted amino acid sequence with known sequences from the databases revealed that the AcMAT1-1-1 protein is most similar to the MAT1-1-1 protein of Gibberella zeae (43.8% amino acid identity in a 226-aa overlap). The predicted DNA-binding motif of the AcMAT1-1-1 protein is very similar to the conserved {alpha}-box domain found in the Saccharomyces cerevisiae Mat{alpha}1p protein and in MAT1-1-1 homologues from other Sordariaceae (Fig. 2A).


Figure 2
View larger version (51K):
[in this window]
[in a new window]

  		FIG. 2. Conserved domains of mating type proteins encoded by the AcMAT1 locus. (A) Multiple-sequence alignment of the {alpha}-box domain region of the AcMAT1-1-1 protein with {alpha}-box domain regions of proteins from other ascomycetes. Abbreviations and accession numbers corresponding to the proteins are as follows: Achr, A. chrysogenum (AM983455); Gzea, Gibberella zeae (Q9HEV7); Gfuj, Gibberella fujikuroi (O93925); Cpur, Claviceps purpurea (BAD72604.1); Pten, Paecilomyces tenuipes (BAC67541); Pans, P. anserina (P35692); Ncra, Neurospora crassa (P19392); Smac, Sordaria macrospora (O42837); Mgri, Magnaporthe grisea (BAC65091.1); and Scer, S. cerevisiae (YP_087100.1). Numbers to the right of the sequences indicate amino acid positions. Shading indicates the degrees of similarity. (B) Multiple-sequence alignment of the HPG domain of the AcMAT1-1-2 protein with HPG mating type proteins from other ascomycetes. The positions of the conserved amino acid residues His, Pro, and Gly are indicated by arrows. For abbreviations, see the legend to panel A. Accession numbers corresponding to the proteins are as follows: A. chrysogenum, AM983456; Gibberella zeae, AAG42811.1; Gibberella fujikuroi, AAC71054.1; Claviceps purpurea, BAD72603.1; Pten, BAC67540; P. anserina, S39889; Neurospora crassa, S65583; Sordaria macrospora, CAA71626.1; and Magnaporthe grisea, BAC65092.1. (C) Multiple-sequence alignment of the HMG domain of the AcMAT1-1-3 protein with MAT1-1-3 HMG mating type proteins from other ascomycetes. For abbreviations, see the legend to panel A. Accession numbers corresponding to the proteins are as follows: A. chrysogenum, AM983457; Gibberella zeae, Q9HEV9; Gibberella fujikuroi, AAC71053.1; Claviceps purpurea, BAD72602.2; P. anserina, Q08143; Neurospora crassa, S65584; and Magnaporthe grisea, BAC65093.2. Amino acid residues conserved in all species are indicated in white with black background shading. Residues conserved in all species with the exception of one or two are indicated in white with gray background shading. Residues conserved in the majority of species are indicated in black with gray background shading.

The AcMAT1-1-2 gene encodes a polypeptide of 331 aa and is not interrupted by introns. The AcMAT1-1-2 protein displayed the highest degree of identity (31.6% amino acid identity in a 335-aa overlap) to the MAT1-1-2 protein containing the conserved HPG domain of Epichloe typhina, an endophytic member of the Clavicipitaceae (Fig. 2B). The coding sequence proposed for the third gene, AcMAT1-1-3, translates into a 190-aa protein and contains three introns of 46, 52, and 48 bp showing typical A. chrysogenum splice sites (30). Comparison of the predicted AcMAT1-1-3 amino acid sequence with known sequences from the databases revealed that AcMAT1-1-3 is most similar to the MAT1-1-3 protein of Ephelis japonica (Clavicipitaceae; 51.6% amino acid identity in a 182-aa overlap). The HMG domain DNA-binding motif is highly similar to HMG domains found in MAT1-1-3 homologues of other sordariomycetes (Fig. 2C). The analysis of the regions upstream and downstream of the mating type genes revealed the presence of an ORF similar to S. cerevisiae APN2 encoding a DNA lyase protein (31), as well as a homologue of the S. cerevisiae SLA2 gene encoding a cytoskeleton assembly protein (28). Frequently, both genes are found close to the MAT loci in filamentous ascomycetes (15, 17, 21, 61).

MAT genes are very useful for phylogenetic analysis due to their high evolution rate (53, 69, 73). We therefore performed an analysis based on the MAT1-1-1 {alpha}-box domain, in order to obtain further information about the phylogenetic position of A. chrysogenum among the ascomycetes. Identical phylogenetic trees were derived using neighbor-joining and parsimony analyses with the {alpha}-box domain of the S. cerevisiae Mat{alpha}1p as the out-group. According to the cladogram shown in Fig. 3, A. chrysogenum shows affiliation with the order Hypocreales and is phylogenetically most closely related to Metarhizium anisopliae.


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 3. Phylogenetic tree of MAT1-1-1 {alpha}-box domains generated by maximum-parsimony and neighbor-joining analyses. Fifty-four amino acid positions were included in the analyses. The tree shown is based on a consensus tree calculated with the PHYLIP programs PROTPARS and NEIGHBOR. The numbers (percentages) indicate the support based on 1,000 replications of the neighbor-joining analysis (numbers to the left of the slashes) and of the maximum-parsimony procedure (numbers to the right of the slashes). Accession numbers corresponding to the analyzed amino acid sequences are as follows: A. chrysogenum, AM983455; Fusarium culmorum, CAD59609.3; Gibberella zeae, A9CCT5; Fusarium poae, CAD59610.3; Gibberella fujikuroi, O93925; Fusarium sacchari, BAE94379.1; Fusarium guttiforme, AAQ18160; Colletotrichum musae, CAD59611.3; Gibberella avenacea, CAD59608.4; Cordyceps takaomontana, BAD95880.1; Cordyceps militaris, BAD72600.1; Verticillium fungicola var. flavidum, BAE93605.1; Tolypocladium inflatum, BAE93602.1; Epichloe typhina, BAD72612.1; Ephelis japonica, BAD72608.1; Claviceps purpurea, BAD72604.1; Metarhizium anisopliae, BAE93598.1; Sordaria macrospora, O42837; Neurospora crassa, P19392; P. anserina, P35692; Chaetomium globosum, XP_001222681.1; Magnaporthe grisea, BAC65091.1; Aspergillus fumigatus, AAX83123.1; Emericella nidulans, Q7Z896; Aspergillus oryzae, Q2U537; Penicillium chrysogenum, AM904544; Penicillium marneffei, Q1A3S7; and S. cerevisiae, YP_087100.1.

Transcriptional expression of mating type gene MAT1-1-3.
Since EST clones of AcMAT1-1-1 and AcMAT1-1-2 were identified in the EST library, both genes appear to be transcriptionally expressed in A. chrysogenum. To determine whether AcMAT1-1-3, the putative third MAT gene, was expressed, RNA was isolated from A. chrysogenum and cDNA was synthesized using AcMAT1-1-3-specific primers. For comparison, cDNAs and amplicons using genomic DNA as the template were generated with primer pairs specific for AcMAT1-1-1, AcMAT1-1-2, and AcMAT1-1-3 (Fig. 4). Sequencing of the AcMAT1-1-3 cDNA confirmed the splicing of the three introns predicted from the genomic DNA, indicating that all three AcMAT genes are transcriptionally active. In addition to the MAT genes, we identified an EST clone corresponding to the APN2 gene; however, we did not identify an EST clone corresponding to the SLA2 gene. The predicted intergenic region between AcMAT1-1-1 and the AcSLA2 gene appeared to be exceptionally large (Fig. 1). Therefore, we used the 4,184-bp sequence of this region in a BLASTN analysis of the A. chrysogenum EST library and identified the 662-bp EST clone AcEST.4.744 corresponding to a transcript of this region. However, the sequence of this transcript has no similarity to any protein or EST clone transcript in the databases.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 4. Transcriptional expression of the genes AcMAT1-1-1, AcMAT1-1-2, and AcMAT1-1-3. Primer pairs as indicated in Materials and Methods were used to generate cDNA or DNA fragments specific for the three ORFs of the AcMAT1-1 locus. RNA transcripts (cDNA) either with (+ RT) or without (– RT) reverse transcriptase were amplified by RT-PCR. gen. DNA, genomic DNA.

Mating type loci in different A. chrysogenum isolates.
To identify a putative MAT1-2 locus and to analyze the distribution of the two mating types, five A. chrysogenum isolates were examined using the {alpha}-box domain sequence-specific AcMAT1-1-1 primers. Only a limited set of six isolates was available from culture collections, including strain ATCC 14553, originally isolated by Giuseppe Brotzu, and the derivative A3/2, which stems from an industrial production line (57). In addition, four other strains collected from at least two different geographic sites (Table 1) were used for our analysis. All strains most probably belong to the same taxon since they contain completely identical internal transcribed spacer sequences (data not shown). Notably, the ITS2 sequence is highly suitable for evaluating whether the taxa of a species can potentially interbreed and has already been used for this purpose for numerous eukaryotes (10). In all strains tested, a 336-bp DNA fragment specific for the AcMAT1-1-1 {alpha}-box mating type gene was present, leading to the conclusion that all A. chrysogenum isolates analyzed are either heterothallic MAT1-1 strains or homothallic strains with a MAT1-2 allele elsewhere in the genome or with only the MAT1-1 locus.

Functional analysis of the AcMAT1-1 locus in P. anserina.
The failure to detect any AcMAT1-2 strains makes it impossible to conduct crossing experiments with strains of the opposite mating type. We therefore performed a functional analysis of the AcMAT1-1 locus with P. anserina as a heterologous host. This heterothallic ascomycete carries a MAT1-1 locus with a high degree of sequence similarity to the AcMAT1-1 locus (Fig. 2). Hence, we transferred cosmid clone P2-G5-hph containing the entire AcMAT1-1 locus into a Pa{Delta}MAT strain (11), yielding the Pa{Delta}MAT-AcMAT1-1ect strain carrying ectopically expressed AcMAT1-1 (Fig. 5A), since it was shown recently that an ectopically expressed MAT locus in this deletion strain confers normal fertility in a cross (11, 51, 75). In contrast, a resident MAT locus was shown previously to interfere with extra copies of MAT locus genes, leading to ascospore lethality (13).


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 5. Rescue of the function of the P. anserina mating type locus by the AcMAT1-1 locus. (A) Flow diagram depicting the origin of strains obtained from the mating (x) of a transformant Pa{Delta}MAT-AcMAT1-1ect (MAT–) strain with the PaMAT1-2 (MAT+) strain. (B) Molecular phenotypes of PaMAT1-2, PaMAT1-1, and Pa{Delta}MAT strains and of the transformant Pa{Delta}MAT-AcMAT1-1ect strain expressing the AcMAT1-1 locus, as well as of the biparental ascospore isolates (bip#1 and bip#2) and 2 of the 28 uniparental ascospore isolates (unip#1 and unip#2). (C) Transcriptional expression of the AcMAT1-1 locus, as well as the PaMAT1-2-1 (FPR1) and PaMAT1-1-1 (FMR1) genes, in P. anserina strains as indicated above the gel. +RT and – RT, with and without reverse transcriptase; gen. DNA, genomic DNA. The sizes of the DNAs and cDNA amplicons are the first and second values, respectively, given at the right margin.

PCR and RT-PCR analyses confirmed that all three AcMAT1-1 genes were integrated and transcriptionally expressed in the P. anserina transformants (Fig. 5B and C). Three individual transformants carrying the transcriptionally expressed AcMAT1-1 locus were then analyzed further by fertilization with spermatia of a PaMAT1-2 (MAT+) strain (Fig. 5A). All three developed further and generated numerous perithecia, indistinguishable from those produced in P. anserina wild-type matings, in crosses with the tester strain (Fig. 6A). However, in most cases, ascus rosettes of perithecia comprised only 3 to 20 asci and the number of asci with fewer than four ascospores was significantly increased compared to the number of such asci produced by the wild type (Fig. 6B). P. anserina has a pseudohomothallic life cycle and develops four-spored asci, in which ascospores contain two nuclei, one of each mating type. A typical binucleate spore is a heterokaryon, i.e., it contains both mating types, and can therefore give rise to a self-fertile mycelium (Fig. 6C). Only a few asci contain five spores, three binucleate and two small uninucleate spores, which produce homokaryons.


Figure 6
View larger version (83K):
[in this window]
[in a new window]

  		FIG. 6. Microscopic investigation of P. anserina strains expressing the AcMAT1-1 locus. (A) Perithecium from a strain obtained from the mating of Pa{Delta}MAT-AcMAT1-1ect (MAT–) and PaMAT1-2 (MAT+) strains. (B) Ascus rosette from the strain shown in panel A. (C) For comparison, an ascus rosette from a strain obtained from a PaMAT1-1 (MAT–) and PaMAT1-2 (MAT+) wild type cross.

To investigate the inheritance of the heterologous MAT locus, 30 randomly isolated ascospores of the fertilized Pa{Delta}MAT-AcMAT1-1ect strain were then tested for simultaneous expression of the endogenous PaMAT1-2-1 (FPR1) gene and the heterologous AcMAT1-1 genes (Fig. 5C). Only 2 of 30 transformants expressed the MAT genes from both loci, while the other 28 seemed to express only the AcMAT1-1 genes (Fig. 5A and C). Thus, these results demonstrate that the AcMAT1-1 locus is transcriptionally expressed and can drive sexual reproduction in P. anserina.

Identification of sex-related genes in A. chrysogenum.
Knowing that AcMAT1-1 is able to carry out fertilization functions in P. anserina, which suggests the potential of A. chrysogenum for sexual reproduction, we then performed in silico analyses to identify sex-related genes in A. chrysogenum. Two different classes of pheromones are known to be involved in sexual reproduction in heterothallic as well as in homothallic filamentous ascomycetes. One class of genes encodes peptide pheromone precursors that contain multiple copies of the mature peptides flanked by protease cleavage sites, while the other class of pheromone genes encodes a small protein with a CAAX motif at the carboxy terminus. This motif is expected to produce a mature lipopeptide pheromone with a C-terminal carboxymethyl isoprenylated cysteine (12, 33, 39). Therefore, to identify putative pheromone precursors encoded by the A. chrysogenum genome, amino acid sequences of the peptide and a lipopeptide pheromone precursor of known fungal pheromone precursors were used as query sequences in TBLASTN searches. No significant hits were found. However, a cDNA encoding a putative pheromone receptor and EST clones encoding putative homologues of proteins involved in the processing of pheromone precursors in S. cerevisiae were identified in the EST library (Table 2).


View this table:
[in this window]
[in a new window]

  		TABLE 2. A. chrysogenum EST clones encoding putative pheromone-processing enzymes

In the yeast S. cerevisiae, after the binding of the pheromones to a cell type-specific receptor, the signal is transmitted by the interaction of a heterotrimeric G protein composed of G{alpha} (Gpa1p), Gβ (Ste4p), and G{gamma} (Ste18p) through a downstream mitogen-activated protein (MAP) kinase cascade encoded by STE20, STE11, STE7, and FUS3. Homologues of all these genes, along with other conserved genes of the S. cerevisiae pheromone response pathway, except FAR1, STE5, KSS1, DIG1, and DIG2, were identified in the A. chrysogenum EST library (Table 3).


View this table:
[in this window]
[in a new window]

  		TABLE 3. A. chrysogenum EST clones encoding putative components of a pheromone response pathway


arrow
DISCUSSION
 
Filamentous ascomycetes lacking any sexual stages have traditionally been placed within the phylum Deuteromycota or have been termed Fungi Imperfecti. This artificial group includes plant and human pathogens, as well as biotechnologically important fungi, such as the β-lactam-producing fungi Penicillium chrysogenum and A. chrysogenum. Recently, however, there has been a proliferation of reports describing the detection of MAT genes and homologues of genes known to be required for sexual pheromone production within species of this group. Thus, there is accumulating evidence suggesting that an increasing number of Fungi Imperfecti have a cryptic sexual cycle and that asexual ascomycetes have the genetic machinery to mate and to develop fertile fruiting bodies, as well as to undergo a full meiotic sexual cycle (3, 21, 26, 32, 50, 67, 76).

A. chrysogenum has a MAT1-1 mating type locus.
In this study, A. chrysogenum strains were shown to carry a MAT1-1 locus structurally similar to MAT loci from heterothallic Sordariales and Hypocreales. The MAT1-1 locus from A. chrysogenum contains at least three genes, AcMAT1-1-1, AcMAT1-1-2, and AcMAT1-1-3, encoding proteins with conserved domains: an {alpha}-box domain, an HPG domain, and an HMG domain, respectively (Fig. 2).

Since we did not succeed in detecting an AcMAT1-2 strain, the exact extension of the MAT1-1 locus cannot be defined. Recently, the sequences in close proximity to MAT loci from diverse ascomycetes were investigated, and it emerged that in an ancestral configuration, MAT loci lie between homologues of S. cerevisiae APN2 and SLA2 (9, 15). Similarly, in A. chrysogenum, APN2 and SLA2 homologues were identified downstream of the AcMAT1-1-3 and AcMAT1-1-1 genes, respectively (Fig. 1). An APN2-SLA2 gene arrangement identical to that in A. chrysogenum is also present in Neurospora crassa, P. anserina, several Gibberella species, Aspergillus fumigatus, and Neosartorya fischeri (15, 21, 61). Hence, according to the configuration of the flanking genes, the AcMAT1-1 locus exhibits a rather ancestral structure.

The genus Acremonium is considered to be a highly polyphyletic genus containing distantly related species (25). Phylogenetic analyses based on partial sequences of the 18S rRNA revealed the affiliation of A. chrysogenum with the order Hypocreales (25). Likewise, the MAT1-1-1 trees obtained from maximum-parsimony and neighbor-joining analyses show a common branching pattern for A. chrysogenum and species of the order Hypocreales and a close relationship to species of the family Clavicipitaceae (Fig. 3). Clavicipitaceous fungi and related anamorphs contain diverse econutritional groups such as pathogens of plants, insects, nematodes, and fungi, as well as endophytes of plants and saprophytes. Divergent groups of the Clavicipitaceae, including the species Cordyceps takaomontana and Cordyceps militaris, are known to lack the mating type gene MAT1-1-3, while more ancient members of this family, e.g., Claviceps purpurea and Metarhizium anisopliae, possess a MAT1-1-3 gene (77). Given that the MAT1-1 locus of A. chrysogenum contains a MAT1-1-3 homologue, it may be considered to be a phylogenetically old member of the Clavicipitaceae family.

In addition to the three conserved MAT genes, a fourth putative ORF corresponding to a 662-bp EST clone, AcEST.4.744, was identified between AcMAT1-1-1 and SLA2. In Neurospora crassa, two genes encoding anonymous transcripts (eat) with mating type-specific size differences were identified in the region surrounding the MAT idiomorphs. One of these genes, eat-2, encodes a protein with a domain highly similar to a domain present in plasma membrane ATPases (58). However, the sequence of AcEST.4.744 bears no similarity to that of either Neurospora crassa eat-2 or eat-1 or to that of any other EST clone in the databases. Due to the lack of an AcMAT1-2 strain, mating type-specific transcript differences could not be analyzed.

Based on the fact that we identified only MAT1-1 strains of A. chrysogenum, we cannot rule out that a MAT1-2 strain does exist. The failure to identify it may be due to the small sample size used in this study. However, at this stage we cannot decide whether A. chrysogenum has a heterothallic or homothallic lifestyle. There are four ways fungi can be homothallic: (i) they can harbor a fused MAT locus comprising both idiomorphs, (ii) they can harbor both MAT alleles at different loci in the genome, (iii) they can switch the mating type locus, or (iv) they can sexually reproduce but carry only one MAT locus (34). The AcMAT1-1 locus appears not to have a MAT1-2-1 homologue, which can be found in MAT loci of homothallic members, either fused or located in close proximity to the {alpha}-box domain gene in Soradiomycetes and Dothidiomycetes (15, 52, 55). Thus, homothallism due to a fused MAT locus can be ruled out. In the genomes of the homothallic Emericella nidulans and Neosartorya fischeri, MAT1-1 and MAT1-2 loci are on separate chromosomes, and in the homothallic Chaetomium globosum, in addition to a MAT1-1 locus, a MAT1-2 locus on another supercontig was identified previously (15, 21, 61). Unfortunately, the genome sequence of A. chrysogenum is still not yet available, and therefore, the possibility of an unlinked second MAT locus encoding a MAT1-2-1 homologue in the A. chrysogenum genome cannot be excluded. It may also be possible that A. chrysogenum has silent MAT loci and can undergo mating type switching, as described previously for several filamentous ascomycetes (14, 34). Interestingly, in some homothallic species of the genus Neurospora (Neurospora africana, Neurospora lineolata, Neurospora galapagonensis, and Neurospora dodgei), only the MAT1-1 locus is present in the genome (23, 24, 53). It has been speculated that in these species, either sexual reproduction does not require the HMG domain protein encoded by the MAT1-2 locus or the function of this protein can be replaced by the MAT1-1-3-encoded HMG protein (40). Alternatively, A. chrysogenum may be a heterothallic fungus with a predominantly homothallic life cycle. Unmated MAT1-1 strains of the heterothallic ascomycete Sordaria brevicollis can form ascospores, whereas MAT1-2 strains are able to form only barren fruiting bodies (59). Thus, the unequal distribution of mating types in A. chrysogenum may be due to a preference for the unisexual mating of MAT1-1 strains. More than 99% of clinical and environmental isolates of the heterothallic basidiomycete Cryptococcus neoformans are MAT{alpha}, and it was shown previously that predominantly haploid MAT{alpha} strains can produce basidiospores by unisexual mating, a modified form of sexual reproduction that involves diploidization and meiosis (36, 37). Quantitative-trait-locus mapping revealed that the MAT{alpha} allele enhances hyphal growth during unisexual mating as a quantitative trait locus and may explain why MAT{alpha} isolates predominate in nature (35).

Sequence analysis of all AcMAT1-1 genes provided no evidence of loss-of-function mutations, indicating that the MAT locus may still contain transcriptionally expressed functional genes that have not been subjected to pseudogene formation, which would be expected to occur if A. chrysogenum had evolved to be asexual. In addition, we showed that A. chrysogenum possesses and transcriptionally expresses some genes crucial for pheromone processing and signal transduction. This finding further supports our hypothesis of a sexual cycle in A. chrysogenum. Homologues of the S. cerevisiae FAR1, STE5, KSS1, DIG1, and DIG2 genes were not identified in the A. chrysogenum EST library. However, since these genes were also absent in Penicillium marneffei, Emericella nidulans, and Aspergillus fumigatus (76), filamentous ascomycetes may use alternatives to the proteins encoded by these genes in pheromone signal processing and transduction.

The MAT1-1 locus of A. chrysogenum induces perithecial and ascospore development in the heterothallic fungus P. anserina.
A functional characterization of the AcMAT genes was carried out by heterologous expression in a Pa{Delta}MAT strain. Interestingly, Pa{Delta}MAT-AcMAT1-1ect strain fungi carrying an ectopic copy of the AcMAT1-1 locus developed fertile perithecia containing rosettes of asci with ascospores (Fig. 6). These results indicate that the AcMAT1-1 locus is not only structurally but also functionally similar to the PaMAT1-1 (MAT–) locus and are consistent with previous findings showing that cloned MAT genes of homothallic and asexual species are functional in heterothallic species (3, 24, 55, 56, 67). Like the asci and ascopores produced by the transgenic strains in this study, asci and ascospores produced by the transgenic Pa{Delta}MAT and Cochliobolus heterostrophus {Delta}MAT strains expressing heterologous MAT genes were found previously to be less abundant than those produced by wild-type strains (3, 51). The AcMAT1-1-1 and AcMAT1-1-3 proteins have highly conserved {alpha}-box domain and HMG box DNA-binding motifs (Fig. 2) and therefore may bind to the same target genes driving fertilization and postfertilization processes. However, interaction with other transcription factors, e.g., MCM1 and STE12, involved in fruiting body development and ascosporogenesis in Sordariales (43, 44) may be affected, thereby altering the transcriptional expression of the target genes and leading to decreased ascus and ascospore production. In our study, RT-PCR analysis revealed that ectopic integrated AcMAT1-1 genes were transcriptionally expressed in P. anserina (Fig. 5C). In contrast, in a previous study, the Aspergillus fumigatus MAT1-2-1 mating type gene ectopically introduced into an Emericella nidulans {Delta}MAT strain was not properly expressed and consequently did not support either fruiting body or ascospore development. The Aspergillus fumigatus MAT1-2-1 gene locus is able to confer full fertility only when it is driven by the Emericella nidulans MAT1-2-1 promoter and integrated by homologous recombination into the Emericella nidulans MAT locus (56).

Interestingly, an analysis of the ascospore progeny produced by the transgenic Pa{Delta}MAT strain showed that only 2 of 30 randomly isolated spores were biparental strains expressing the MAT genes from both loci. All other spore isolates were uniparental strains expressing only the AcMAT1-1 genes (Fig. 5). The production of uniparental progeny by P. anserina can often be observed among strains with a mutated MAT1-1-3 (SMR2) gene. In this situation, the genetic markers of the uniparental progeny come exclusively from the parent containing the mutation. Viable ascospores are formed after a haploid meiosis event during which the random segregation of chromosomes results by chance in even distribution (70). One of the mutations leading to uniparental progeny is smr2(E202A) (4, 70). The analysis of an amino acid alignment of the AcMAT1-1-3 and PaMAT1-1-3 (SMR2) proteins revealed that the amino acid residue E202 in PaMAT1-1-3 corresponds to A148 in AcMAT1-1-3. In P. anserina, the wild-type MAT1-1-3 (SMR2) protein seems to repress MAT1-2 (MAT+) fertilization functions in MAT1-1 (MAT–) sexual organs by interaction with MAT1-1-1 (FMR1). According to the internuclear recognition model (70), nuclei carrying a mutated MAT1-1-3 (SMR2) gene express both the MAT1-1 and MAT1-2 functions required for internuclear recognition and trigger self-recognition.

Together, the identification of conserved components of the pheromone response pathway and the functional conservation of the MAT genes provide the first evidence to suggest a sexual cycle in A. chrysogenum. To date, there are no reports on possible reasons for the presence of functional MAT genes in supposedly asexual filamentous fungi. However, one explanation is that some of the supposedly asexual species may have a cryptic sexual cycle (8). Another possibility is that sexual reproduction is a rare event that is hard to detect (74) and thus remains to be described. Nevertheless, the activation of a sexual cycle in A. chrysogenum would increase options for alternative strain improvement strategies.

Further studies are needed to search the A. chrysogenum genome for other sequences known to be necessary for meiotic recombination in other fungi and then to examine their function. To clarify whether the reproductive mode of A. chrysogenum is heterothallic or homothallic, the A. chrysogenum genome should also be further analyzed for the presence of a MAT1-2-1 mating type gene homologue. Since all A. chrysogenum strains analyzed in this study were derived from type culture collections, we cannot exclude the possibility that they may have lost fertility during the long time of storage. Thus, future studies will examine A. chrysogenum strains isolated directly from the environment for past or ongoing meiotic recombination. Finally, whether mating type proteins in A. chrysogenum regulate other genes not directly involved in sexual reproduction remains to be elucidated.


arrow
ACKNOWLEDGMENTS
 
We thank Ingeborg Godehardt, Kerstin Kalkreuter, Stefanie Mertens, and Susanne Schlewinski for excellent technical assistance and Hubert Kürnsteiner, Ernst Friedlin, Thomas Specht, and Ivo Zadra (Kundl, Austria) for the A. chrysogenum EST sequences and for fruitful discussion. We are grateful to Evelyne Coppin (Institut de Genetique et Microbiologie, CNRS-Université Paris Sud, Orsay, France) for providing the Pa{Delta}MAT strain.

We acknowledge financial support from Sandoz GmbH (Kundl, Austria) and from the Christian Doppler Society (Vienna, Austria).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany. Phone: 49 (0)234 3226212. Fax: 49 (0)234 3214184. E-mail: ulrich.kueck{at}rub.de Back

{triangledown} Published ahead of print on 8 August 2008. Back

{ddagger} These authors contributed equally to this work. Back


arrow
REFERENCES
 
    1
  1. Abraham, E. 1990. Selective reminiscences of β-lactam antibiotics: early research on penicillin and cephalosporins. Bioessays 12:601-606.[CrossRef][Medline]
    2. 2
  2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
    3. 3
  3. Arie, T., I. Kaneko, T. Yoshida, M. Noguchi, Y. Nomura, and I. Yamaguchi. 2000. Mating-type genes from asexual phytopathogenic ascomycetes Fusarium oxysporum and Alternaria alternata. Mol. Plant-Microbe Interact. 13:1330-1339.[Medline]
    4. 4
  4. Arnaise, S., D. Zickler, S. Le Bilcot, C. Poisier, and R. Debuchy. 2001. Mutations in mating-type genes of the heterothallic fungus Podospora anserina lead to self-fertility. Genetics 159:545-556.[Abstract/Free Full Text]
    5. 5
  5. Barber, S. M., U. Giesecke, A. Reichert, and W. Minas. 2004. Industrial enzymatic production of cephalosporin-based β-lactams. Adv. Biochem. Eng. Biotechnol. 88:179-215.[Medline]
    6. 6
  6. Brotzu, G. 1948. Ricerche su di un nuovo antibiotico. Lav. Inst. Igiene Cagliari 1948:1-11.
    7. 7
  7. Bullock, W., J. Fernandez, and J. Short. 1987. XL-1 Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5:376-378.
    8. 8
  8. Burt, A., D. A. Carter, G. L. Koenig, T. J. White, and J. W. Taylor. 1996. Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis. Proc. Natl. Acad. Sci. USA 93:770-773.[Abstract/Free Full Text]
    9. 9
  9. Butler, G., C. Kenny, A. Fagan, C. Kurischko, C. Gaillardin, and K. H. Wolfe. 2004. Evolution of the MAT locus and its Ho endonuclease in yeast species. Proc. Natl. Acad. Sci. USA 101:1632-1637.[Abstract/Free Full Text]
    10. 10
  10. Coleman, A. W. 2007. Pan-eukaryote ITS2 homologies revealed by RNA secondary structure. Nucleic Acids Res. 35:3322-3329.[Abstract/Free Full Text]
    11. 11
  11. Coppin, E., S. Arnaise, V. Contamine, and M. Picard. 1993. Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation. Mol. Gen. Genet. 241:409-414.[Medline]
    12. 12
  12. Coppin, E., C. de Renty, and R. Debuchy. 2005. The function of the coding sequences for the putative pheromone precursors in Podospora anserina is restricted to fertilization. Eukaryot. Cell 4:407-420.[Abstract/Free Full Text]
    13. 13
  13. Coppin, E., and R. Debuchy. 2000. Co-expression of the mating-type genes involved in internuclear recognition is lethal in Podospora anserina. Genetics 155:657-669.[Abstract/Free Full Text]
    14. 14
  14. Coppin, E., R. Debuchy, S. Arnaise, and M. Picard. 1997. Mating type and sexual development in ascomycetes. Microbiol. Mol. Biol. Rev. 61:411-428.[Abstract]
    15. 15
  15. Debuchy, R., and B. G. Turgeon. 2006. Mating-type structure, evolution, and function in euascomycetes, p. 293-323. In U. Kües and R. Fischer (ed.), The Mycota, vol. I. Growth, differentiation and sexuality. Springer, Berlin, Germany.
    16. 16
  16. Dreyer, J., H. Eichhorn, E. Friedlin, H. Kürnsteiner, and U. Kück. 2007. A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum. Appl. Environ. Microbiol. 73:3412-3422.[Abstract/Free Full Text]
    17. 17
  17. Dyer, P. S. 2007. Sexual reproduction and significance of MAT in the aspergilli, p. 123-142. In J. Heitman, J. W. Kronstad, J. W. Taylor, and L. A. Casselton (ed.), Sex in fungi: molecular determination and evolutionary implications. ASM Press, Washington, DC.
    18. 18
  18. Esser, K. 1974. Podospora anserina, p. 531-551. In P. C. King (ed.), Handbook of genetics. Plenum Press, New York, NY.
    19. 19
  19. Felsenstein, J. 2004. PHYLIP: Phylogeny Inference Package, version 3.6. Department of Genome Sciences and Department of Biology, University of Washington, Seattle.
    20. 20
  20. Fraser, J. A., and J. Heitman. 2004. Evolution of fungal sex chromosomes. Mol. Microbiol. 51:299-306.[CrossRef][Medline]
    21. 21
  21. Galagan, J. E., S. E. Calvo, C. Cuomo, L. J. Ma, J. R. Wortman, S. Batzoglou, S. I. Lee, M. Bastürkmen, C. C. Spevak, J. Clutterbuck, V. Kapitonov, J. Jurka, C. Scazzocchio, M. Farman, J. Butler, S. Purcell, S. Harris, G. H. Braus, O. Draht, S. Busch, C. D'Enfert, C. Bouchier, G. H. Goldman, D. Bell-Pedersen, S. Griffiths-Jones, J. H. Doonan, J. Yu, K. Vienken, A. Pain, M. Freitag, E. U. Selker, D. B. Archer, M. A. Peñalva, B. R. Oakley, M. Momany, T. Tanaka, T. Kumagai, K. Asai, M. Machida, W. C. Nierman, D. W. Denning, M. Caddick, M. Hynes, M. Paoletti, R. Fischer, B. Miller, P. Dyer, M. S. Sachs, S. A. Osmani, and B. W. Birren. 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105-1115.[CrossRef][Medline]
    22. 22
  22. Gams, W. 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes). Gustav Fischer Verlag, Stuttgart, Germany.
    23. 23
  23. Glass, N. L., R. L. Metzenberg, and N. B. Raju. 1990. Homothallic Sordariaceae from nature: the absence of strains containing only the a mating type sequence. Exp. Mycol. 14:274-289.[CrossRef]
    24. 24
  24. Glass, N. L., and M. L. Smith. 1994. Structure and function of a mating-type gene from the homothallic species Neurospora africana. Mol. Gen. Genet. 244:401-409.[Medline]
    25. 25
  25. Glenn, A. E., C. W. Bacon, R. Price, and R. T. Hanlin. 1996. Molecular phylogeny of Acremonium and its taxonomic implications. Mycologia 88:369-383.[CrossRef]
    26. 26
  26. Hoff, B., S. Pöggeler, and U. Kück. 2008. Eighty years after its discovery, Fleming's Penicillium strain discloses the secret of its sex. Eukaryot. Cell 7:465-470.[Abstract/Free Full Text]
    27. 27
  27. Hoff, B., E. K. Schmitt, and U. Kück. 2005. CPCR1, but not its interacting transcription factor AcFKH1, controls fungal arthrospore formation in Acremonium chrysogenum. Mol. Microbiol. 56:1220-1233.[CrossRef][Medline]
    28. 28
  28. Holtzman, D. A., S. Yang, and D. G. Drubin. 1993. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J. Cell Biol. 122:635-644.[Abstract/Free Full Text]
    29. 29
  29. Janus, D., B. Hoff, E. Hofmann, and U. Kück. 2007. An efficient fungal RNA-silencing system using the DsRed reporter gene. Appl. Environ. Microbiol. 73:962-970.[Abstract/Free Full Text]
    30. 30
  30. Jekosch, K., and U. Kück. 1999. Codon bias in the β-lactam producer Acremonium chrysogenum. Fungal Genet. Newsl. 46:11-13.
    31. 31
  31. Johnson, R. E., C. A. Torres-Ramos, T. Izumi, S. Mitra, S. Prakash, and L. Prakash. 1998. Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites. Genes Dev. 12:3137-3143.[Abstract/Free Full Text]
    32. 32
  32. Kerényi, Z., A. Moretti, C. Waalwijk, B. Oláh, and L. Hornok. 2004. Mating type sequences in asexually reproducing Fusarium species. Appl. Environ. Microbiol. 70:4419-4423.[Abstract/Free Full Text]
    33. 33
  33. Kim, H., and K. A. Borkovich. 2006. Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa. Eukaryot. Cell 5:544-554.[Abstract/Free Full Text]
    34. 34
  34. Lin, X., and J. Heitman. 2007. Mechanisms of homothallism in fungi and transitions between heterothallism and homothallism, p. 35-57. In J. Heitman, J. W. Kronstad, J. W. Taylor, and L. A. Casselton (ed.), Sex in fungi: molecular determination and evolutionary implications. ASM Press, Washington, DC.
    35. 35
  35. Lin, X., J. C. Huang, T. G. Mitchell, and J. Heitman. 2006. Virulence attributes and hyphal growth of C. neoformans are quantitative traits and the MAT{alpha} allele enhances filamentation. PLoS Genet. 2:e187.[CrossRef][Medline]
    36. 36
  36. Lin, X., C. M. Hull, and J. Heitman. 2005. Sexual reproduction between partners of the same mating type in Cryptococcus neoformans. Nature 434:1017-1021.[CrossRef][Medline]
    37. 37
  37. Lin, X., A. P. Litvintseva, K. Nielsen, S. Patel, A. Floyd, T. G. Mitchell, and J. Heitman. 2007. {alpha}AD{alpha} hybrids of Cryptococcus neoformans: evidence of same-sex mating in nature and hybrid fitness. PLoS Genet. 3:1975-1990.[Medline]
    38. 38
  38. Martín, J. F., R. V. Ullán, and J. Casqueiro. 2004. Novel genes involved in cephalosporin biosynthesis: the three-component isopenicillin N epimerase system. Adv. Biochem. Eng. Biotechnol. 88:91-109.[Medline]
    39. 39
  39. Mayrhofer, S., J. M. Weber, and S. Pöggeler. 2006. Pheromones and pheromone receptors are required for proper sexual development in the homothallic ascomycete Sordaria macrospora. Genetics 172:1521-1533.[Abstract/Free Full Text]
    40. 40
  40. Metzenberg, R. L., and N. L. Glass. 1990. Mating type and mating strategies in Neurospora. Bioessays 12:53-59.[CrossRef][Medline]
    41. 41
  41. Minuth, W., P. Tudzynski, and K. Esser. 1982. Extrachromosomal genetics of Cephalosporium acremonium. Curr. Genet. 5:227-231.[CrossRef]
    42. 42
  42. Nash, C. H., and F. M. Huber. 1971. Antibiotic synthesis and morphological differentiation of Cephalosporium acremonium. Appl. Microbiol. 22:6-10.[Medline]
    43. 43
  43. Nolting, N., and S. Pöggeler. 2006. A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora. Eukaryot. Cell 5:1043-1056.[Abstract/Free Full Text]
    44. 44
  44. Nolting, N., and S. Pöggeler. 2006. A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis. Mol. Microbiol. 62:853-868.[CrossRef][Medline]
    45. 45
  45. Nowrousian, M., S. Masloff, S. Pöggeler, and U. Kück. 1999. Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity. Mol. Cell. Biol. 19:450-460.[Abstract/Free Full Text]
    46. 46
  46. Nowrousian, M., C. Ringelberg, J. C. Dunlap, J. J. Loros, and U. Kück. 2005. Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus Sordaria macrospora. Mol. Gen. Genomics 273:137-149.[CrossRef][Medline]
    47. 47
  47. Osiewacz, H. D., A. Skaletz, and K. Esser. 1991. Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes. Appl. Microbiol. Biotechnol. 35:38-45.[Medline]
    48. 48
  48. Page, R. D. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.[Free Full Text]
    49. 49
  49. Paoletti, M., C. Rydholm, E. U. Schwier, M. J. Anderson, G. Szakacs, F. Lutzoni, J. P. Debeaupuis, J. P. Latgé, D. W. Denning, and P. S. Dyer. 2005. Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus. Curr. Biol. 15:1242-1248.[CrossRef][Medline]
    50. 50
  50. Pöggeler, S. 2002. Genomic evidence for mating abilities in the asexual pathogen Aspergillus fumigatus. Curr. Genet. 42:153-160.[CrossRef][Medline]
    51. 51
  51. Pöggeler, S. 2007. MAT and its role in the homothallic ascomycete Sordaria macrospora, p. 171-188. In J. Heitman, J. W. Kronstad, J. W. Taylor, and L. A. Casselton (ed.), Sex in fungi: molecular determination and evolutionary implications. ASM Press, Washington, DC.
    52. 52
  52. Pöggeler, S. 2001. Mating-type genes for classical strain improvements of ascomycetes. Appl. Microbiol. Biotechnol. 56:589-601.[CrossRef][Medline]
    53. 53
  53. Pöggeler, S. 1999. Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes. Curr. Genet. 36:222-231.[CrossRef][Medline]
    54. 54
  54. Pöggeler, S., M. Nowrousian, S. Jacobsen, and U. Kück. 1997. An efficient procedure to isolate fungal genes from an indexed cosmid library. J. Microbiol. Methods 29:49-61.[CrossRef]
    55. 55
  55. Pöggeler, S., S. Risch, U. Kück, and H. D. Osiewacz. 1997. Mating-type genes from the homothallic fungus Sordaria macrospora are functionally expressed in a heterothallic ascomycete. Genetics 147:567-580.[Abstract]
    56. 56
  56. Pyrzak, W., K. Y. Miller, and B. L. Miller. 2008. The mating type protein Mat1-2 from asexual Aspergillus fumigatus drives sexual reproduction in fertile Aspergillus nidulans. Eukaryot. Cell 7:1029-1040.[Abstract/Free Full Text]
    57. 57
  57. Radzio, R., and U. Kück. 1997. Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 48:58-65.[CrossRef][Medline]
    58. 58
  58. Randall, T. A., and R. L. Metzenberg. 1998. The mating type locus of Neurospora crassa: identification of an adjacent gene and characterization of transcripts surrounding the idiomorphs. Mol. Gen. Genet. 259:615-621.[CrossRef][Medline]
    59. 59
  59. Robertson, S. J., D. J. Bond, and N. D. Read. 1998. Homothallism and heterothallism in Sordaria brevicollis. Mycol. Res. 102:1215-1223.[CrossRef]
    60. 60
  60. Rodríguez-Sáiz, M., M. Lembo, L. Bertetti, R. Muraca, J. Velasco, A. Malcangi, J. L. de la Fuente, and J. L. Barredo. 2004. Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker. FEMS Microbiol. Lett. 235:43-49.[Medline]
    61. 61
  61. Rydholm, C., P. S. Dyer, and F. Lutzoni. 2007. DNA sequence characterization and molecular evolution of MAT1 and MAT2 mating-type loci of the self-compatible ascomycete mold Neosartorya fischeri. Eukaryot. Cell 6:868-874.[Abstract/Free Full Text]
    62. 62
  62. Sambrook, J., and D. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    63. 63
  63. Schmitt, E. K., B. Hoff, and U. Kück. 2004. AcFKH1, a novel member of the forkhead family, associates with the RFX transcription factor CPCR1 in the cephalosporin C-producing fungus Acremonium chrysogenum. Gene 342:269-281.[CrossRef][Medline]
    64. 64
  64. Schmitt, E. K., B. Hoff, and U. Kück. 2004. Regulation of cephalosporin biosynthesis. Adv. Biochem. Eng. Biotechnol. 88:1-43.[Medline]
    65. 65
  65. Schmitt, E. K., R. Kempken, and U. Kück. 2001. Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum: specific DNA-protein interactions and characterization of the transcription factor PACC. Mol. Genet. Genomics 265:508-518.[CrossRef][Medline]
    66. 66
  66. Schmitt, E. K., and U. Kück. 2000. The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors. J. Biol. Chem. 31:9348-9357.
    67. 67
  67. Sharon, A., K. Yamaguchi, S. Christiansen, B. A. Horwitz, O. C. Yoder, and B. G. Turgeon. 1996. An asexual fungus has the potential for sexual development. Mol. Gen. Genet. 251:60-68.[Medline]
    68. 68
  68. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
    69. 69
  69. Turgeon, B. G. 1998. Application of mating type gene technology to problems in fungal biology. Annu. Rev. Phytopathol. 36:115-137.[CrossRef][Medline]
    70. 70
  70. Turgeon, B. G., and R. Debuchy. 2007. Cochliobolus and Podospora: mechanisms of sex determination and the evolution of reproductive lifestyle, p. 93-121. In J. Heitman, J. W. Kronstad, J. W. Taylor, and L. A. Casselton (ed.), Sex in fungi: molecular determination and evolutionary implications. ASM Press, Washington, DC.
    71. 71
  71. Turgeon, B. G., and O. C. Yoder. 2000. Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genet. Biol. 31:1-5.[CrossRef][Medline]
    72. 72
  72. Velasco, J., J. Luis Adrio, M. Angel Moreno, B. Díez, G. Soler, and J. L. Barredo. 2000. Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum. Nat. Biotechnol. 18:857-861.[CrossRef][Medline]
    73. 73
  73. Voigt, K., A. J. Cozijnsen, J. Kroymann, S. Pöggeler, and B. J. Howlett. 2005. Phylogenetic relationships between members of the crucifer pathogenic Leptosphaeria maculans species complex as shown by mating type (MAT1-2), actin, and beta-tubulin sequences. Mol. Phylogenet. Evol. 37:541-557.[CrossRef][Medline]
    74. 74
  74. Ware, S. B., E. C. Verstappen, J. Breeden, J. R. Cavaletto, S. B. Goodwin, C. Waalwijk, P. W. Crous, and G. H. Kema. 2007. Discovery of a functional Mycosphaerella teleomorph in the presumed asexual barley pathogen Septoria passerinii. Fungal Genet. Biol. 44:389-397.[CrossRef][Medline]
    75. 75
  75. Wirsel, S., B. G. Turgeon, and O. C. Yoder. 1996. Deletion of the Cochliobolus heterostrophus mating-type (MAT) locus promotes the function of MAT transgenes. Curr. Genet. 29:241-249.[Medline]
    76. 76
  76. Woo, P. C., K. T. Chong, H. Tse, J. J. Cai, C. C. Lau, A. C. Zhou, S. K. Lau, and K. Y. Yuen. 2006. Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei. FEBS Lett. 580:3409-3416.[CrossRef][Medline]
    77. 77
  77. Yokoyama, E., M. Arakawa, K. Yamagishi, and A. Hara. 2006. Phylogenetic and structural analyses of the mating-type loci in Clavicipitaceae. FEMS Microbiol. Lett. 264:182-191.[CrossRef][Medline]
    78. 78
  78. Yokoyama, E., K. Yamagishi, and A. Hara. 2005. Heterothallism in Cordyceps takaomontana. FEMS Microbiol. Lett. 250:145-150.[CrossRef][Medline]
    79. 79
  79. Yokoyama, E., K. Yamagishi, and A. Hara. 2003. Structures of the mating-type loci of Cordyceps takaomontana. Appl. Environ. Microbiol. 69:5019-5022.[Abstract/Free Full Text]
    80. 80
  80. Yun, S. H., M. L. Berbee, O. C. Yoder, and B. G. Turgeon. 1999. Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors. Proc. Natl. Acad. Sci. USA 96:5592-5597.[Abstract/Free Full Text]

Applied and Environmental Microbiology, October 2008, p. 6006-6016, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01188-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.
Agricola
Right arrow Articles by Pöggeler, S.
Right arrow Articles by Kück, U.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»