Combination of Proteogenomics with Peptide De Novo Sequencing Identifies New Genes and Hidden Posttranscriptional Modifications.

Proteogenomics combines proteomics, genomics, and transcriptomics and has considerably improved genome annotation in poorly investigated phylogenetic groups for which homology information is lacking. Furthermore, it can be advantageous when reinvestigating well-annotated genomes. Here, we applied an advanced proteogenomics approach, combining standard proteogenomics with peptide de novo sequencing, to refine annotation of the well-studied model fungus Sordaria macrospora We investigated samples from different developmental and physiological conditions, resulting in the detection of 104 so-far hidden proteins and annotation changes in 575 genes, including 389 splice site refinements. Significantly, our approach provides peptide-level evidence for 113 single-amino-acid variations and 15 C-terminal protein elongations originating from A-to-I RNA editing, a phenomenon recently detected in fungi. Coexpression and phylostratigraphic analysis of the refined proteome suggest that new functions in evolutionarily young genes correlate with distinct developmental stages. In conclusion, our advanced proteogenomics approach supports and promotes functional studies of fungal model systems.IMPORTANCE Next-generation sequencing techniques have considerably increased the number of completely sequenced eukaryotic genomes. These genomes are mostly automatically annotated, and ab initio gene prediction is commonly combined with homology-based search approaches and often supported by transcriptomic data. The latter in particular improve the prediction of intron splice sites and untranslated regions. However, correct prediction of translation initiation sites (TIS), alternative splice junctions, and protein-coding potential remains challenging. Here, we present an advanced proteogenomics approach, namely, the combination of proteogenomics and de novo peptide sequencing analysis, in conjunction with Blast2GO and phylostratigraphy. Using the model fungus Sordaria macrospora as an example, we provide a comprehensive view of the proteome that not only increases the functional understanding of this multicellular organism at different developmental stages but also immensely enhances the genome annotation quality.

Tools for advanced and targeted genetic manipulation of the ß-lactam antibiotic producer Acremonium chrysogenum.

Acremonium chrysogenum is the major producer of the ß-lactam antibiotic cephalosporin C and therefore of great importance for the pharmaceutical industry. However, this filamentous fungus is known to reproduce solely by asexual means, shows only sporadic conidiospore production, and has gradual fragmentation of the vegetative mycelium into arthrospores. Due to these peculiar growth characteristics and life style, strain improvement by recombinant technologies is much more challenging than for other biotechnologically relevant fungi. Here, we describe several molecular tools for genetic engineering of A. chrysogenum, including a ΔAcku70 deletion strain for homologous recombination. No physiological or morphological changes occurred due to deletion of the ku70 gene or integration of the nat1 cassette in this recipient strain. We also used a xylose-inducible promoter from Sordaria macrospora (Smxyl) to demonstrate induction of the gfp reporter gene in A. chrysogenum. The Smxyl promoter was used for construction of a vector molecule to develop a one-step FLP/FRT recombination system in A. chrysogenum. This system was then used in the ΔAcku70 deletion strain to construct a marker-free recipient strain for targeted DNA insertion into genomic DNA. The applicability of our tools was demonstrated by construction of a marker-free transgenic strain, lacking any foreign genes.

Microarray and real-time PCR analyses reveal mating type-dependent gene expression in a homothallic fungus.

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The DeltaSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up- or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from DeltaSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500-fold down-regulated in the DeltaSmta-1 mutant while in all other sterile mutants this gene is up-regulated.

Development of molecular tools for the mulundocandin producer Aspergillus sydowii: DNA-mediated transformation and reporter gene expression.

The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.

Identification of transcriptionally expressed pheromone receptor genes in filamentous ascomycetes.

Detection of pheromone genes in filamentous ascomycetes implicated the presence of pheromone receptor genes. Similar to yeasts and basidiomycetes, these might be involved in a G-protein triggered signal transduction pathway during mating. We have identified two pheromone receptor genes, named pre1 and pre2, in the genome of the heterothallic filamentous ascomycete Neurospora crassa and the closely related homothallic Sordaria macrospora. The deduced pre1 gene product is a putative seven-transmembrane protein, which displays a high-level amino acid identity with the a-factor receptor Ste3p of Saccharomyces cerevisiae, and is also homologous to lipopeptide pheromone receptors of basidiomycetes. The deduced pre2 product displays significant sequence similarity with the S. cerevisiae STE2 gene product, the alpha-factor receptor. Pair-wise comparisons between pheromone receptor genes of N. crassa and S. macrospora revealed an extremely low degree of nucleotide conservation in these genes, suggesting that they evolved very rapidly. The two genes are transcriptionally expressed in both N. crassa and S. macrospora. Northern and reverse transcription-polymerase chain reaction analyses indicate that in N. crassa, expression of the receptor genes does not occur in a mating type specific manner. Thus, filamentous ascomycetes appear to posses and express pheromone receptor genes.

Functional analysis of promoter sequences of cephalosporin C biosynthesis genes from Acremonium chrysogenum: specific DNA-protein interactions and characterization of the transcription factor PACC.

An analysis of the pcbC promoter from the cephalosporin C-producing filamentous fungus Acremonium chrysogenum was performed using fungal transformants carrying reporter gene fusions. By investigating sequential deletion derivatives of the pcbC promoter region, a DNA fragment was identified, which is responsible for transcriptional activation of the pcbC gene. Sequence analysis of this fragment revealed a consensus binding site for the fungal transcription factor PACC. Gel-retardation experiments with crude extracts of A. chrysogenum confirmed the specific binding of a protein to the PACC binding site. The subsequent cloning of the pacC homolog from A. chrysogenum allowed the identification of an ORF of 621 amino acids encoded by four exons. The polypeptide shows about 35% sequence identity to other fungal PACC proteins. A PACC protein fragment synthesized in E. coli was used for in vitro binding assays, and specific binding of the zinc-finger transcription factor to its consensus binding sites in the promoter regions of four cephalosporin C biosynthesis genes could be demonstrated. The bi-directional promoters of the genes pcbAB-pcbC and cefEF-cefG contain two PACC binding sites each. The data obtained strongly suggest that, in A. chrysogenum, the zinc-finger transcription factor PACC is involved in the transcriptional regulation of the genes involved in cephalosporin C biosynthesis.

Intron-specific RNA binding proteins in the chloroplast of the green alga Chlamydomonas reinhardtii.

Mitochondria and chloroplasts both contain group II introns which are believed to be the ancestors of nuclear spliceosomal introns. We used the mitochondrial group II intron rI1 from the green alga Scenedesmus obliquus for biochemical characterization of intron-specific RNA binding proteins. rI1 is correctly spliced from a chloroplast precursor RNA when integrated into the chloroplast genome of Chlamydomonas reinhardtii. Glycerol gradients revealed the sedimentation profile of transcripts containing intron rI1 in native C. reinhardtii extracts and in deproteinized RNA preparations, thus indicating the association of rI1 containing transcripts with high molecular weight ribonucleoprotein complexes in vivo. Furthermore, the specific binding of a 61 kDa protein and a 31 kDa protein with the conserved domain IV was demonstrated using a set of intron derivatives for in vitro RNA binding experiments. We propose that we have biochemically characterized 'general splicing factors', which enable the successful splicing even of mitochondrial introns in chloroplasts.

Loss of glucose repression in an Acremonium chrysogenum beta-lactam producer strain and its restoration by multiple copies of the cre1 gene.

In the filamentous fungus Acremonium chrysogenum, biosynthesis of the beta-lactam antibiotic cephalosporin C is repressed by glucose. A wild-type strain grown in the presence of glucose shows a reduction of transcripts derived from the pcbC and cefEF biosynthesis genes. Interestingly, the amount of the pcbC transcript is not affected in another strain with enhanced cephalosporin C production, suggesting a correlation between strain improvement and deregulation of glucose repression. The function of the glucose repressor CRE1 in this regulation was further analyzed by transforming both A. chrysogenum strains with multiple copies of the cre1 gene. The molecular analysis of transformants revealed that additional copies of the cre1 gene restore wild-type-like regulation of pcbC gene expression in the semi-producer strain, while repression of the cefEF gene expression is increased. Overall, our data indicate a regulation of the pcbC and the cefEF gene expression by CRE1.

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon.

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42781bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT-PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

Glucose dependent transcriptional expression of the cre1 gene in Acremonium chrysogenum strains showing different levels of cephalosporin C production.

The cre1 gene from the beta-lactam producer Acremonium chrysogenum has been isolated and characterized in order to study glucose-dependent gene expression in this biotechnically important fungus. The deduced protein sequence is highly similar to amino-acid sequences of other known glucose repressors from filamentous fungi, and carries conserved zinc-finger and regulatory motifs. Contrary to cre gene expression in Trichoderma reesei and Aspergillus nidulans, the transcript level of the cre1 gene from an A. chrysogenum wild-type strain is increased in the presence of glucose. Remarkably, the glucose-dependent transcriptional upregulation does not take place in another A. chysogenum strain, which displays enhanced production of the beta-lactam antibiotic cephalosporin C. We surmise that the de-regulation of cre1 is connected with the increased production rate in this strain.

The unicellular green alga Chlamydomonas reinhardtii as an experimental system to study chloroplast RNA metabolism.

Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

The fungal acl1 and acl2 genes encode two polypeptides with homology to the N- and C-terminal parts of the animal ATP citrate lyase polypeptide.

ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl-CoA, which is mainly used for the biosynthesis of fatty acids and sterols. In this paper, we show for the first time that in filamentous fungi two different subunits of ACL are encoded by two separate genes. This is in contrast to animals where ACL is encoded by a single gene. Data are presented on acl genes from the filamentous fungi Sordaria macrospora and Gibberella pulicaris. In S. macrospora, both genes, acl1 and acl2, are clustered within a region of 10 kb and are divergently transcribed.

Comparative analysis of the mating-type loci from Neurospora crassa and Sordaria macrospora: identification of novel transcribed ORFs.

The mating-type locus controls mating and sexual development in filamentous ascomycetes. In the heterothallic ascomycete Neurospora crassa, the genes that confer mating behavior comprise dissimilar DNA sequences (idiomorphs) in the mat a and mat A mating partners. In the homothallic fungus Sordaria macrospora, sequences corresponding to both idiomorphs are located contiguously in the mating-type locus, which contains one chimeric gene, Smt A-3, that includes sequences which are similar to sequences found at the mat A and mat a mating-type idiomorphs in N. crassa. In this study, we describe the comparative transcriptional analysis of the chimeric mating-type region of S. macrospora and the corresponding region of the N. crassa mat a idiomorph. By means of RT-PCR experiments, we identified novel intervening sequences in the mating-type loci of both ascomycetes and, hence, concluded that an additional ORF, encoding a putative polypeptide of 79 amino acids, is present in the N. crassa mat a idiomorph. Furthermore, our analysis revealed co-transcription of the novel gene with the mat a-1 gene in N. crassa. The same mode of transcription was found in the corresponding mating-type region of S. macrospora, where the chimeric Smt A-3 gene is co-transcribed with the mat a-specific Smt a-1 gene. Analysis of a Smt A-3 cDNA revealed optional splicing of two introns. We believe that this is the first report of co-transcription of protein-encoding nuclear genes in filamentous fungi. Possible functions of the novel ORFs in regulating mating-type gene expression are discussed.

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless.

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented.

The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors.

Here we report the isolation and characterization of a novel transcription factor from the cephalosporin C-producing fungus Acremonium chrysogenum. We have identified a protein binding site in the promoter of the beta-lactam biosynthesis gene pcbC, located 418 nucleotides upstream of the translational start. Using the yeast one-hybrid system, we succeeded in isolating a cDNA clone encoding a polypeptide, which binds specifically to the pcbC promoter. The polypeptid shows significant sequence homology to human transcription factors of the regulatory factor X (RFX) family and was designated CPCR1. A high degree of CPCR1 binding specificity was observed in in vivo and in vitro experiments using mutated versions of the DNA binding site. The A. chrysogenum RFX protein CPCR1 recognizes an imperfect palindrome, which resembles binding sites of human RFX transcription factors. One- and two-hybrid experiments with truncated versions of CPCR1 showed that the protein forms a DNA binding homodimer. Nondenaturing electrophoresis revealed that the CPCR1 protein exists in vitro solely in a multimeric, probably dimeric, state. Finally, we isolated a homologue of the cpcR1 gene from the penicillin-producing fungus Penicillium chrysogenum and determined about 60% identical amino acid residues in the DNA binding domain of both fungal RFX proteins, which show an overall amino acid sequence identity of 29%.

The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.

During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.

Identification of DNA sequences controlling light- and chloroplast-dependent expression of the lhcb1 gene from Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, expression of the lhcb1 gene encoding a chlorophyll-a/b-binding protein of photosystem II is highly regulated by light, inhibitors of chlorophyll synthesis, as well as by circadian rhythms. In light/dark synchronized cultures, the rapid increase of lhcb1 mRNA levels during the light phase is regulated primarily at the transcriptional level. We have used the arylsulphatase (ars) reporter gene to analyze the lhcb1 5' upstream sequences for the presence of light-responsive elements. In transformants carrying chimeric reporter genes, accumulation of lhcb1/ars mRNA is markedly stimulated by light, with a time course similar to that of transcripts from the endogenous lhcb1 gene. Promoter deletion studies revealed that a 255-bp fragment of the lhcb1 5' upstream region is sufficient to confer proper light regulation on the promoterless ars gene. Moreover, the region between positions -255 and -122 with respect to the start site of translation were found to contain one or more light-responsive elements. Strikingly, these sequences also seem to be involved in chloroplast-dependent lhcb1 gene expression as indicated by Northern analyses of transformants with photo-oxidatively damaged chloroplasts. This suggests that both light- and chloroplast-dependent expression of the lhcb1 gene are mediated by the same cis-acting elements.

Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity.

During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.