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Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.

The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (–) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (–) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (–) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

Genetic analysis of all Mucor-like fungi is severely impaired by the low efficiency of transformation systems and the genetic instability of the introduced plasmid constructs. The transformation efficiency of one of the model systems among mucoralean fungi, Absidia glauca, was improved considerably by microprojectile bombardment. For this purpose, a plasmid was constructed conferring (i) neomycin resistance as a selective marker and (ii) fluorescence due to expression of the gfpgene from the jellyfish Aequorea victoria. Compared with previous techniques, this method offers increased efficiency, with considerably easier handling than procedures based on protoplasts and, therefore, improved reliability. The uninucleate sporangiospores of A. glaucacan be transformed early during the germination process. At this stage the number of nuclei ranges between 1 and 2. Thus, the abundance of transgenic nuclei in the coenocytic mycelia is high, and fewer problems are encountered with detecting low expression levels of the genes used for selection and monitoring of transformants.

The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitroamplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.

The randomly amplified polymorphic DNA technique (RAPD) is a modification of PCR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population- or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/ Maxam) and chain termination (Sänger) techniques. Based on this sequence information, highly specific oligonucleo- tide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculons.

Methods for isolation and electrophoretic analysis of surface proteins from the mucoraceous fungus Absidia glaucaare described. By analysing the set of cell wall associated proteins which can be extracted with 1 M LiCl, we could show marked differences between submersed and aerial mycelium. Furthermore, during differentiation of the aerial mycelium protein patterns change remarkably. We were able to demonstrate the existence of mating type specific proteins, the most prominent of which is a 15 kDa polypeptide expressed in submersed as well as in aerial hyphae of the (+) mating type.

Methods for isolation and electrophoretic analysis of surface proteins from the mucoraceous fungus Absidia glaucaare described. By analysing the set of cell wall associated proteins which can be extracted with 1 M LiCl, we could show marked differences between submersed and aerial mycelium. Furthermore, during differentiation of the aerial mycelium protein patterns change remarkably. We were able to demonstrate the existence of mating type specific proteins, the most prominent of which is a 15 kDa polypeptide expressed in submersed as well as in aerial hyphae of the (+) mating type.

A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neor) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neor phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.

The infection of the model organism Absidia glauca by P. parasitica is accompanied by the fusion of both mycelia. By two lines of evidence we were able to show that this process is associated with the transfer of genes. First, auxotrophically labelled A. glauca mutants are efficiently complemented as a consequence of transfer of the parasite's genetic material. Second, for a plasmidcoded dominant marker (neomycin resistance), which is expressed in either organism, we proved the presence of plasmid DNA in recombinant recipients by molecular analysis at the DNA level. We propose the term para-recombinants for describing recombinant inter-generic chimærae, which are generated as a consequence of mycoparasitism.

A small protein with a molecular mass of 15 kDa, which is specifically found on the hyphal surface of a (+) mating-type strain of the model zygomycete Absidia glauca, was purified to electrophoretic homogeneity and partially sequenced. The corresponding gene was cloned by means of an oligonucleotide probe deduced from the protein sequence. It could be localized on an extrachromosomal circular DNA element with a total length of 1250 bp. Electron microscopic analysis of A. glauca DNA showed that small extrachromosomal DNAs with varying length are a common feature of this zygomycete. There are no indications of additional chromosomal copies of the gene for this surface protein, and the plasmid is absent from DNA preparations of the (-) mating type. The copy number ranges around three per haploid genome, and a single transcript with a length of 400 bp, coding for the surface protein, could be found by employing a hybridization probe which spans the complete fungal plasmid. This is the first report of naturally occurring extrachromosomal DNA in a Mucor-like fungus, and the only example where an integral protein of the cell wall is encoded by a plasmid.

In addition to conventional methods for the identification of fungi, molecular techniques at the DNA level are increasingly being employed. In order to check the validity of such experimental approaches, we have analyzed the well-defined species Parasitella parasitica, which belongs to the family Mucoraceae (Mucorales, Zygometes). The seven strains of this species, which are available from international strain collections, were analyzed by several molecular methods: restriction fragment length polymorphism analysis (RFLP), the random primer-dependent polymerase chain reaction (RAPD-PCR), and electrophoretic karyotyping. Unexpectedly, these strains are highly diverse at the molecular level. By these techniques they can be divided consistently into two different groups. Nevertheless, all seven strains belong to a single species. They show no morphological differences and sexual spores (zygospores) were found in all possible combinations either within or between the two groups. Southern-blot analysis of genomic DNA of all P. parasitica strains with RAPD-PCR-derived labelled probes shows the existence of repetitive elements characteristic for only one group of P. parasitica. In addition, chromosome sizes, which were separated by rotating-field electrophoresis, were highly divergent, and ranged from 3 to 6.5 Mb in one group and between 2 and 4.5 Mb in the other. The RAPD-PCR patterns also discriminate both groups of P. parasitica. However, they are very similar if strains of a single group are compared. Therefore, we propose that the determination of fungal species by molecular techniques should be vetted at least by morphological and physiological parameters and, whenever possible, by mating experiments.

Experimental conditions for the separation of chromosomes from the model zygomycete Absidia glauca by rotating field electrophoresis were established. The sexually compatible strains of the mating type pair A. glauca CBS 100.48 (+) and CBS 101.48 (-) showed considerable differences in their electrophoretic karyotype. By Southern hybridization with homologous probes we have mapped the chromosomal locations for rDNA, the repetitive element rAg1, and the genes for actin and the elongation factor EF1. For the mapping of ubiquitin information we used a heterologous probe from U. maydis. The combination of electrophoretic karyotyping and Southern mapping proved to be a useful tool for characterizing mutant genotypes, which were induced by integrative transformation.

Total cell DNA of Absidia glauca has a GC-content of 44.6% ± 0.5% as determined from optical melting profiles which is in good accordance with values from equilibrium centrifugation in bisbenzimide containing CsCl gradients (46.2% = 1.1%), whereas mitochondrial DNA has a GC-content of only 30%. The genome size of Absidia glauca is approximately 36,000 kb, 8.6 times that of Escherichia coli. Three kinetically different fractions could be identified in reassociation experiments: a foldback-DNA fraction, comprising approximately 10% of the total DNA, repetitive DNA (25%) and single copy DNA (65%). This relatively high amount of repetitive DNA could partly be ascribed to ribosomal DNA (13%) and a new interspersed repetitive element (“rAg1”) which has been cloned in pBR325.

We have cloned fragments from mitochondrial and chromosomal DNA of the zygomycete Absidia glauca in Saccharomyces cerevisiae using the ARS selection vector YIp5. Though it has not been possible to select ARS elements from chromosomal DNA, we succeeded in isolating two clones of mitochondrial origin that support autonomous replication in bakers' yeast. DNA from these plasmids has been shown to hybridize with mitochondrial DNA from both mating types. Generation times of the transformed yeast strain in selective medium are around 20 h. In liquid minimal medium only 6% of the cells contain the plasmid; in complete medium a mitotic stability of 50% has been determined.

Protoplasts of auxotrophic mutants of the heterothallic zygomycete Absidia glauca were efficiently fused by electrofusion. Fusion heterokaryons between the complementary mating types, which grew prototrophically, were selected. Among 72 fusion colonies which were further analysed, 11 did not only grow prototrophically but also combined both mating types as they formed zygotes within the same mycelium. These homothallic hybrids possessed two sets of rDNA repeats originating from the complementary mating types. Those fusion colonies which gave only a (+) type mating reaction, synthesized a 15 kDa (+) type-specific surface protein; fusion colonies which mated as (-) type, did not produce this surface protein. In homothallic fusion products no expression of the (+) type-specific protein could be detected. Single spore derivatives of fusion colonies in general show the phenotype of only one fusion partner, but in some cases prototrophic recombinants were obtained. This system opens up the possibility for genetic analysis of an organism with inefficient meiotic system, lacking a natural parasexual cycle.

Experimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.

A DNA fragment of mitochondrial origin from the mucoraceous fungus Absidia glauca promoting autonomous plasmid replication in Saccharomyces cerevisiae was sequenced and functionally characterised. We could show that the original mitochondrial insert cloned in Yip5 contains two regions with ARS activity which mutually inhibit each other. All plasmid derivatives replicating in yeast are rapidly lost during growth under non-selective conditions. In addition to one ARS consensus sequence with only one base substitution, the mitochondrial insert contains 18 related sequences with two base pair exchanges. With one exception all consensus sequences are preceded by sequence motifs strongly resembling ARS boxes.

Methods for obtaining the release, regeneration, and fusion of protoplasts from the dioecious zygomycete Absidia glauca are presented. Somatic hybridization was obtained between protoplasts from several amino acid auxotrophic mutants either belonging to the same or complementary mating type. Both kinds of mating type information are expressed within the same organism and give rise to a monoecious phenotype. Without the selective pressure on the auxotrophic markers used for the construction of the fusion strains, the ability to form zygospores is rapidly lost. However, the gene clusters for ribosomal RNA which are distinguishable between mating types are stable and maintained in the fusion strains, independent of growth conditions.

A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts. Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate. The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis. No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.

Millions of humans and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which exclusively infect keratinized host structures. To provide broad insights into the molecular basis of the pathogenicity-associated traits, we report the first genome sequences of two closely phylogenetically related dermatophytes, Arthroderma benhamiae and Trichophyton verrucosum, both of which induce highly inflammatory infections in humans.

A cDNA clone for maize endosperm sucrose synthetase of 620 nucleotide pairs length was obtained by cloning double stranded DNA obtained from the total maize endosperm poly(A) RNA in pBR322, and identifying the appropriate clone by hybrid-promoted translation. In Southern blotting to genomic BamHI-digested DNA, a single band only of ∼20 Kb lights up, indicating that the sucrose synthetase gene is unique, or that closely linked copies are located on this DNA fragment.