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PubMed2004

A functional screen identifies lateral transfer of beta-glucuronidase (gus) from bacteria to fungi.

Lateral gene transfer (LGT) from prokaryotes to microbial eukaryotes is usually detected by chance through genome-sequencing projects. Here, we explore a different, hypothesis-driven approach. We show that the fitness advantage associated with the transferred gene, typically invoked only in retrospect, can be used to design a functional screen capable of identifying postulated LGT cases. We hypothesized that beta-glucuronidase (gus) genes may be prone to LGT from bacteria to fungi (thought to lack gus) because this would enable fungi to utilize glucuronides in vertebrate urine as a carbon source. Using an enrichment procedure based on a glucose-releasing glucuronide analog (cellobiouronic acid), we isolated two gus(+) ascomycete fungi from soils (Penicillium canescens and Scopulariopsis sp.). A phylogenetic analysis suggested that their gus genes, as well as the gus genes identified in genomic sequences of the ascomycetes Aspergillus nidulans and Gibberella zeae, had been introgressed laterally from high-GC gram(+) bacteria. Two such bacteria (Arthrobacter spp.), isolated together with the gus(+) fungi, appeared to be the descendants of a bacterial donor organism from which gus had been transferred to fungi. This scenario was independently supported by similar substrate affinities of the encoded beta-glucuronidases, the absence of introns from fungal gus genes, and the similarity between the signal peptide-encoding 5' extensions of some fungal gus genes and the Arthrobacter sequences upstream of gus. Differences in the sequences of the fungal 5' extensions suggested at least two separate introgression events after the divergence of the two main Euascomycete classes. We suggest that deposition of glucuronides on soils as a result of the colonization of land by vertebrates may have favored LGT of gus from bacteria to fungi in soils.

Amino Acid Sequence↗
PubMed2006

A class III histidine kinase acts as a novel virulence factor in Botrytis cinerea.

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.

Blotting, Southern↗
PubMed1997

A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: description of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov.

A phylogenetic investigation of the ascomycetous yeast genus Saccharomyces was performed by using 18S rRNA gene sequence analysis. Comparative sequence analysis showed that the genus is phylogenetically very heterogeneous. Saccharomyces species were found to be phylogenetically interdispersed with members of other ascomycetous genera (e.g., the genera Kluyveromyces, Torulaspora, and Zygosaccharomyces). The four species of the Saccharomyces sensu stricto complex (viz., Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus) were found to be phylogenetically closely related to one another, displaying exceptionally high levels of sequence similarity (> or = 99.9%). These four species formed a natural group that was quite separate from the other Saccharomyces and non-Saccharomyces species examined. Saccharomyces exiguus and its anamorph, Candida holmii, were found to be genealogically almost identical and, along with Saccharomyces barnettii, formed a stable group closely related to, but nevertheless distinct from, Kluyveromyces africanus, Kluyveromyces lodderae, Saccharomyces rosinii, Saccharomyces spencerorum, and Saccharomyces sp. strain CBS 7662T (T = type strain). Saccharomyces spencerorum and Kluyveromyces lodderae displayed a particularly close genealogical affinity with each other, as did Saccharomyces castellii and Saccharomyces dairensis. Similarly, Saccharomyces servazzii, Saccharomyces unisporus, and Saccharomyces sp. strain CBS 6904 were found to be genotypically highly related and to form a phylogenetically distinct lineage. The recently reinstated species Saccharomyces transvaalensis was found to form a distinct lineage and displayed no specific association with any other Saccharomyces or non-Saccharomyces species. Saccharomyces kluyveri formed a very loose association with a group which included Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, and Zygosaccharomyces fermentati. Saccharomyces spp. strain CBS 6334T, on the other hand, displayed no specific association with any of the other Saccharomyces spp. studied, although a neighbor-joining analysis did reveal that this strain exhibited a loose phylogenetic affinity with Kluyveromyces polysporus and Kluyveromyces yarrowii. On the basis of the phylogenetic findings, two new Saccharomyces species, Saccharomyces kunashirensis (with type strain CBS 7662) and Saccharomyces martiniae (with type strain CBS 6334), are described.

DNA, Fungal↗
PubMed1979

Taxonomic significance of yeast sphaeroplast release after enzymatic treatment of intact cells.

Treatment of whole yeast cells with a mixture of a reducing agent and 1,3-beta-glucanase isolated from Basidiomycete QM806 led to the production of sphaeroplasts from ascomycetes, from some fungi imperfecti, but not from basidiomycetes. Association of 1,3-beta-glucanase with a second enzyme, 1,4-alpha-glucanase, from Trichoderma viride, was required for sphaeroplast release from some, but not all, basidiomycetes and fungi imperfecti. The ability of yeast cells to liberate sphaeroplasts following appropriate enzymic treatment is proposed as a taxonomic criterion for differentiating basidiomycetous from ascomycetous yeasts and for classifying fungi imperfecti yeasts.

Ascomycota↗
PubMed1991

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences.

The classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.

Base Sequence↗
PubMed2000

Two distinct 18S rRNA secondary structures in Dipodascus (Hemiascomycetes).

The nucleotide sequences of the 18S rRNA gene from ascomycetous yeast-like fungi in the genera Dipodascus, Galactomyces and Geotrichum were determined and the tested strains were separated into two groups by sequence length. In group 1, the length and secondary structure of 18S rRNA corresponded to those of typical eukaryotes. In group 2, the 18S rRNA gene sequences were about 150 nt shorter than those of most other eukaryotes and the predicted secondary structure lacked helices 10 and E21-5. Many substitutions and some deletions in group 2 18S rRNA gene were not only found in variable regions, but also in regions that are highly conserved among ascomycetes. Despite the considerable differences in 18S rRNA gene sequence and secondary structure between group 2 and other fungi, including group 1, phylogenetic analysis revealed that groups 1 and 2 are closely related. These findings suggest that a number of deletions occurred in the 18S rRNA of the common ancestor of group 2 strains.

Ascomycota↗
PubMed2003

Candida kunwiensis sp. nov., a yeast associated with flowers and bumblebees.

A novel asexual ascomycetous yeast, Candida kunwiensis (SG99-26T = KCTC 17041T = CBS 9067T), was isolated from sweet potato (Ipomoea batatas) flowers in Korea and from the body surface of pollinating bumblebees in Germany. Comparative analysis of the D1/D2 domain of 26S rDNA of all available sequences for ascomycetous yeasts showed that the novel species was phylogenetically related to the genus Metschnikowia, but the sequence similarity was low. Morphologically and physiologically, C. kunwiensis in many ways resembles Metschnikowia pulcherrima, but can be distinguished from this species by its ability to assimilate lactic acid and its inability to produce pulcherrimin.

Animals↗
PubMed2003

Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts.

Expressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1,700 5' end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E<10(-5)) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best BLAST match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade.

Animals↗
PubMed2003

Cryptons: a group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi.

A new group of transposable elements, which the authors have named cryptons, was detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. These elements are unlike any previously described transposons. An archetypal member of the group, crypton Cn1, is 4 kb in length and is present at a low but variable copy number in a variety of C. neoformans strains. It displays interstrain variations in its insertion sites, suggesting recent mobility. The internal region contains a long gene, interrupted by several introns. The product of this gene contains a putative tyrosine recombinase near its middle, and a region similar in sequence to the DNA-binding domains of several fungal transcription factors near its C-terminus. The element contains no long repeat sequences, but is bordered by short direct repeats which may have been produced by its insertion into the host genome by recombination. Many of the structural features of crypton Cn1 are conserved in the other known cryptons, suggesting that these elements represent the functional forms. The presence of cryptons in ascomycetes and basidiomycetes suggests that this is an ancient group of elements (>400 million years old). Sequence comparisons suggest that cryptons may be related to the DIRS1 and Ngaro1 groups of tyrosine-recombinase-encoding retrotransposons.

Amino Acid Sequence↗
PubMed1993

ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

Ascomycota↗
PubMed2005

Hyperdiversity of ectomycorrhizal fungus assemblages on oak seedlings in mixed forests in the southern Appalachian Mountains.

Diversity of ectotrophic mycobionts on outplanted seedlings of two oak species (Quercus rubra and Quercus prinus) was estimated at two sites in mature mixed forests in the southern Appalachian Mountains by sequencing nuclear 5.8S rRNA genes and the flanking internal transcribed spacer regions I and II (ITS). The seedlings captured a high diversity of mycorrhizal ITS-types and late-stage fungi were well represented. Total richness was 75 types, with 42 types having a frequency of only one. The first and second order jackknife estimates were 116 and 143 types, respectively. Among Basidiomycetes, tomentelloid/thelephoroid, russuloid, and cortinarioid groups were the richest. The ascomycete Cenococcum geophilum was ubiquitously present. Dominant fungi included a putative Tuber sp. (Ascomycetes), and Basidiomycetes including a putative Craterellus sp., and Laccaria cf. laccata. Diversity was lower at a drier high elevation oak forest site compared to a low elevation mesic cove--hardwood forest site. Fungal specificity for red oak vs. white oak seedlings was unresolved. The high degree of rarity in this system imposes limitations on the power of community analyses at finer scales. The high mycobiont diversity highlights the potential for seedlings to acquire carbon from mycelial networks and confirms the utility of using outplanted seedlings to estimate ectomycorrhizal diversity.

Altitude↗
PubMed2006

The Aspergillus nidulans F-box protein GrrA links SCF activity to meiosis.

Cellular differentiation relies on precise and controlled means of gene expression that act on several levels to ensure a flexible and defined spatio-temporal expression of a given gene product. In our aim to identify transcripts enriched during fruiting body formation of the homothallic ascomycete Aspergillus (Emericella) nidulans, the grrA gene could be identified in a negative subtraction hybridization screening procedure. It encodes a protein similar to fungal F-box proteins, which function as substrate receptors for ubiquitin ligases, and that is highly related to the Saccharomyces cerevisiae regulatory protein Grr1p. Expression studies confirmed induction of grrA transcription and expression of its gene product during cleistothecial development of A. nidulans. Functional complementation of a yeast grr1Delta mutant was achieved by overexpression of the grrA coding sequence. A grrADelta deletion mutant resembles the wild-type in hyphal growth, asexual sporulation, Hülle cell formation or development of asci-containing cleistothecia, but is unable to produce mature ascospores due to a block in meiosis as demonstrated by cytological staining of cleistothecial contents. Our results specify a particular involvement of the E3 ubiquitin ligase SCFGrrA in meiosis and sexual spore formation of an ascomyceteous fungus and shed light on the diverse functions of ubiquitin-proteasome-mediated protein degradation in eukaryotic development.

Amino Acid Sequence↗
PubMed2006

Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador.

Cavendishia nobilis var. capitata is an endemic member of the Ericaceae growing as a hemiepiphyte in the tropical mountain rain forest of southern Ecuador. Mycorrhizas were collected from 20 individuals along an altitudinal gradient between 1850 and 2300 m. Transmission electron microscopy was used to study the symbiotic association in detail, and phylogenetic analyses based on nuclear rDNA coding for the ribosomal large subunit (nucLSU) were carried out to identify the associated mycorrhizal fungi. Microscopic and ultrastructural investigations showed the formation of a hyphal sheath, intercellular penetration of fine hyphae and colonization of the cortical cells by swollen hyphae of the same fungus. These structures were formed by hymenomycetes and ascomycetes. Molecular phylogenetic analysis detected seven groups of mycorrhizal fungi belonging to the Sebacinales. This is the first study to obtain evidence of ectendomycorrhizas in the Vaccinioideae. The ascomycetous nucLSU sequences belonged to members of the Leotiomycetes. The ectendomycorrhiza of C. nobilis with Sebacinales is discussed as a specific, hitherto undescribed mycorrhizal subcategory of ectomycorrhizas. We propose the term 'cavendishioid mycorrhiza'. This subcategory is most likely specific for the Andean clade of Ericaceae.

Ecosystem↗
PubMed2006

In vitro decomposition of Sphagnum by some microfungi resembles white rot of wood.

The abilities of some ascomycetes (Myxotrichaceae) from a Sphagnum bog in Alberta to degrade cellulose, phenolics, and Sphagnum tissue were compared with those of two basidiomycetes. Most Myxotrichaceae degraded cellulose and tannic acid, and removed cell-wall components simultaneously from Sphagnum tissues, whereas the basidiomycetes degraded cellulose and insoluble phenolics, and preferentially removed the polyphenolic matrix from Sphagnum cell walls. Mass losses from Sphagnum varied from up to 50% for some ascomycetes to a maximum of 35% for the basidiomycetes. The decomposition of Sphagnum by the Myxotrichaceae was analogous to the white rot of wood and indicates that these fungi have the potential to cause significant mineralization of carbon in bogs.

Ascomycota↗
PubMed2006

Comparative gene expression analysis of fruiting body development in two filamentous fungi.

The ascomycete Pyronema confluens is a member of the Pezizales and readily forms fruiting bodies under laboratory conditions. Here, we report the first molecular analysis of fruiting body development in this filamentous fungus. Two P. confluens cDNA libraries were generated, one derived from mycelium undergoing sexual development, and the other from vegetative mycelium. From each library, 96 clones were end-sequenced, resulting in the identification of 132 different genes. Expression studies of 10 P. confluens genes by quantitative real-time PCR identified seven genes that are transcriptionally up- or down regulated during sexual development when compared with vegetative growth. As a first step towards a comparison of gene expression during fruiting body development in different filamentous fungi, transcript levels of the corresponding homologs from the distantly related ascomycete Sordaria macrospora were analyzed by quantitative real-time PCR. The analyses revealed similar expression patterns during sexual development for several of the S. macrospora genes when compared with P. confluens.

Ascomycota↗
PubMed1995

Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny.

Phylogenetic hypotheses provide a context for examining the evolution of heterotrophic lifestyles. The lichen lifestyle, which is the symbiotic association of fungi with algae, is found in various representatives of Dicaryomycotina, both Ascomycetes and Basidiomycetes. A highly resolved parsimony analysis of small subunit ribosomal DNA (SSU rDNA) sequences suggests at least five independent origins of the lichen habit in disparate groups of Ascomycetes and Basidiomycetes. Because lichen associations arose from parasitic, mycorrhizal, or free-living saprobic fungi, neither mutualism nor parasitism should be construed as endpoints in symbiont evolution.

Ascomycota↗
PubMed2000

Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad. Ectomycorrhizal mycelium.

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5' and 3' ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.

Ascomycota↗
PubMed2003

Dynamics of bacterial and fungal communities on decaying salt marsh grass.

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of Spartina alterniflora detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the alpha-Proteobacteria and one with the Cytophagales) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the alpha-Proteobacteria and two ascomycete fungi (Phaeosphaeria spartinicola and environmental isolate "4clt").

Alphaproteobacteria↗