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Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii.

Genetic divergence at the SODA (manganese-dependent superoxide dismutase, MnSOD) locus were compared in six Pneumocystis carinii formae speciales isolated from mouse, rabbit, human, macaque and pig. A degenerate oligonucleotide primer strategy was designed to amplify 85-90% of the full-length SODA gene from P. carinii genomic DNA isolates. DNA sequence analysis revealed an A/T bias in the nucleotide composition (71-77.2%) and the presence of seven small introns (41-142 bp), interrupting each P. carinii open reading frame (ORF) at the same position. The MnSOD deduced amino acid sequences from all P. carinii isolates shared residues which were conserved within the MnSOD family and which are required for enzymatic activity and binding of the cofactor metal. Phylogenetic analysis including MnSOD sequences from representatives of the fungal phyla Basidiomycota and Ascomycota indicated that the P. carinii formae speciales form a monophyletic group that is related to the budding yeasts (subphylum Saccharomycotina, previously called class Hemiascomycetes) in the Ascomycota. In the whole Pneumocystis group, P. carinii f. sp. hominis, P. carinii f. sp. macacae and P. carinii f. sp. oryctolagi MnSOD sequences clustered together, as did the rat-derived P. carinii and P. carinii f. sp. muris sequences.

Amino Acid Sequence↗

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire.

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Carbohydrate Metabolism↗

Distinct difference in the polyamine compositions of bryophyta and pteridophyta.

Polyamine contents of various species of plants and fungi including Bryophyta, Pteridophyta, Gymnospermae, Ascomycota, Basidiomycota, and Lichenobionta were determined by the combination of six chromatographic techniques. Polyamines examined included putrescine, spermidine, spermine, 1,3-diaminopropane (diaminopropane), sym-norspermidine (norspermidine), sym-norspermine (norspermine), thermospermine, caldopentamine, homocaldopentamine, cadaverine, aminopropylcadaverine, sym-homospermidine (homospermidine), agmatine, and canavalmine. In addition to the widely occurring polyamines (putrescine, spermidine, and spermine), the "unusual" polyamines norspermidine and norspermine were found to be widely distributed in Bryophyta and Lichenobionta. These two polyamines were not detected in any species of Pteridophyta, Gymnospermae, and fungi even though their possible precursor, diaminopropane, was found in some species. Homospermidine was one of the major polyamines in Bryophyta and Lichenobionta, and was detected in most species of Pteridophyta and sporadically in higher plants. Agmatine was detected in most species of Bryophyta and in certain species of Gymnospermae. These data suggest that norspermidine, norspermine, and homospermidine can serve as chemical phylogenic and taxonomic markers in Plantae and Fungi.

Fungi↗

Symbiotic fungal associations in 'lower' land plants.

An analysis of the current state of knowledge of symbiotic fungal associations in 'lower' plants is provided. Three fungal phyla, the Zygomycota, Ascomycota and Basidiomycota, are involved in forming these associations, each producing a distinctive suite of structural features in well-defined groups of 'lower' plants. Among the 'lower' plants only mosses and Equisetum appear to lack one or other of these types of association. The salient features of the symbioses produced by each fungal group are described and the relationships between these associations and those formed by the same or related fungi in 'higher' plants are discussed. Particular consideration is given to the question of the extent to which root fungus associations in 'lower' plants are analogous to 'mycorrhizas' of 'higher' plants and the need for analysis of the functional attributes of these symbioses is stressed. Zygomycetous fungi colonize a wide range of extant lower land plants (hornworts, many hepatics, lycopods, Ophioglossales, Psilotales and Gleicheniaceae), where they often produce structures analogous to those seen in the vesicular-arbuscular (VA) mycorrhizas of higher plants, which are formed by members of the order Glomales. A preponderance of associations of this kind is in accordance with palaeohbotanical and molecular evidence indicating that glomalean fungi produced the archetypal symbioses with the first plants to emerge on to land. It is shown, probably for the first time, that glomalean fungi forming typical VA mycorrhiza with a higher plant (Plantago lanceolata) can colonize a thalloid liverwort (Pellia epiphylla), producing arbuscules and vesicles in the hepatic. The extent to which these associations, which are structurally analogous to mycorrhizas, have similar functions remains to be evaluated. Ascomycetous associations are found in a relatively small number of families of leafy liverworts. The structural features of the fungal colonization of rhizoids and underground axes of these plants are similar to those seen in mycorrhizal associations of ericaceous plants like Vaccinium. Cross inoculation experiments have confirmed that a typical mycorrhizal endophyte of ericaceous plants, Hymenoscyphus ericae, will form associations in liverworts which are structurally identical to those seen in nature. Again, the functional significance of these associations remains to be examined. Some members of the Jungermanniales and Metzgeriales form associations with basidiomycetous fungi. These produce intracellular coils of hyphae, which are similar to the pelotons seen in orchid mycorrhizas, which also involve basidiomycetes. The fungal associates of the autotrophic Aneura and of its heterotrophic relative Cryptothallus mirabilis have been isolated. In the latter case it has been shown that the fungal symbiont is an ectomycorrhizal associate of Betula, suggesting that the apparently obligate nature of the association between the hepatic and Betula in nature is based upon requirement for this particular heterotroph.

Fungi↗

Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains.

A comparative study of the V4, V6 and V9 domains of the mitochondrial small-subunit (SSU) rRNA was conducted to evaluate the use of these sequences to investigate phylogenetic relatedness within the genus Pleurotus. The PCR products encompassing these regions from 48 isolates belonging to 16 Pleurotus species were sequenced and compared. From this comparison, the length and sequence of the three domains were found to be constant within a species. Significant inter-species variations due to insertion/deletion events were found, in most cases occurring in regions not directly involved in the maintainance of the standard SSU rRNA secondary structure. Phylogenetic analysis based upon these mitochondrial sequences was in agreement with relationships previously established by morphological descriptions and with previous studies based upon the nuclear genome or isozymes; moreover such analysis resolved some ambiguities in earlier analyses. It was confirmed that P. ostreatus and P. florida represent a single species, as well as P. pulmonarius and P. sajor-caju. The phylogenetic analysis also made it possible to assess the relative positions of P. rattenburyi, P. lampas, P. sapidus, P. colombinus and P. eryngii. The results clearly showed that sequences of the V4, V6 and V9 domains of the mitochondrial SSU rRNA could provide good markers for use in the taxonomy and phylogeny of species of Basidiomycota. Because of their nucleotide conservation, the major advantage of these species-specific markers was the possibility to study only one isolate from each species to determine phylogenetic relatedness.

Base Sequence↗

Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence.

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited beta-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Amino Acid Sequence↗

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants.

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.

Adaptation, Physiological↗

5S ribosomal RNA sequence of Pneumocystis carinii and its phylogenetic association with "Rhizopoda/Myxomycota/Zygomycota group".

The cytoplasmic 5S ribosomal RNA sequence from Pneumocystis carinii was determined and compared with those of 382 eukaryotes and an evolutionary tree was constructed to establish the phylogenetic position of Pneumocystis. The data suggest that Pneumocystis is associated with the Rhizopoda/Myxomycota/Zygomycota group but not with common fungi, such as Ascomycota or Basidiomycota, nor with other protozoa.

Animals↗

Yeast diversity sampling on the San Juan Islands reveals no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale.

Biological diversity has been estimated for various phyla of life, such as insects and mammals, but in the microbe world is has been difficult to determine species richness and abundance. Here we describe a study of species diversity of fungi with a yeast-like colony morphology from the San Juan Islands, a group of islands that lies southeast of Vancouver Island, Canada. Our sampling revealed that the San Juan archipelago biosphere contains a diverse range of such fungi predominantly belonging to the Basidiomycota, particularly of the order Tremellales. One member of this group, Cryptococcus gattii, is the etiological agent of a current and ongoing outbreak of cryptococcosis on nearby Vancouver Island. Our sampling did not, however, reveal this species. While the lack of recovery of C. gattii does not preclude its presence on the San Juan Islands, our results suggest that the Strait of Juan de Fuca may be serving as a geographical barrier to restrict the dispersal of this primary human fungal pathogen into the United States.

British Columbia↗

Discovery of synthetic penaeidin activity against antibiotic-resistant fungi.

Penaeidins are antimicrobial peptides from shrimp that are constituted by divergent classes of peptide isoforms in an individual organism. Penaeidin sequence variation suggests functional diversity in the host and promises differential activities if applied to treat infections in humans. We have synthesized isoform 4 of penaeidin class 3 from the Atlantic shrimp, Litopenaeus setiferus, by native ligation using three peptide segments. Our synthesis approach led to the discovery of an irreversible side reaction that was successfully suppressed, a discovery, which has particular relevance to the synthesis of cysteine-rich peptides. The antimicrobial activity of full-length penaeidin and the N-terminal proline-rich domain of this isoform were compared with the corresponding peptides of penaeidin class 4 isoform 1 using a wide range of bacteria and fungi. New aspects of penaeidin function are reported that include activity against fungi of the phylum Basidiomycota (Cryptococcus strains), activity against fungi that are pathogenic to humans and effectiveness in the context of antibiotic resistance mechanisms (Cryptococcus and Candida spp.). The proline-rich domain of penaeidin class 4 shows the highest relative antimicrobial activity, while exhibiting no cytotoxicity to human monocytes, and therefore stands out as a potential peptide therapeutic.

Amino Acid Sequence↗

Glomalean fungi from the Ordovician.

Fossilized fungal hyphae and spores from the Ordovician of Wisconsin (with an age of about 460 million years) strongly resemble modern arbuscular mycorrhizal fungi (Glomales, Zygomycetes). These fossils indicate that Glomales-like fungi were present at a time when the land flora most likely only consisted of plants on the bryophytic level. Thus, these fungi may have played a crucial role in facilitating the colonization of land by plants, and the fossils support molecular estimates of fungal phylogeny that place the origin of the major groups of terrestrial fungi (Ascomycota, Basidiomycota, and Glomales) around 600 million years ago.

Biological Evolution↗

Head-to-head comparison of the activities of currently available antifungal agents against 3,378 Spanish clinical isolates of yeasts and filamentous fungi.

We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 microg/ml for amphotericin B, 0.29 microg/ml for flucytosine, 0.97 microg/ml for fluconazole, 0.07 microg/ml for itraconazole, 0.04 microg/ml for voriconazole, 0.15 microg/ml for caspofungin, and 0.03 microg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 microg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

Amphotericin B↗

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

DNA Primers↗

Piriformospora indica, a cultivable plant-growth-promoting root endophyte

Piriformospora indica (Hymenomycetes, Basidiomycota) is a newly described cultivable endophyte that colonizes roots. Inoculation with the fungus and application of fungal culture filtrate promotes plant growth and biomass production. Due to its ease of culture, this fungus provides a model organism for the study of beneficial plant-microbe interactions and a new tool for improving plant production systems.

Journal Article↗

Oligonucleotide fingerprinting of rRNA genes for analysis of fungal community composition.

Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones derived from soil generated 455 clusters. A pairwise sequence analysis showed that clones with average sequence identities of 99.2% were grouped into the same cluster. To examine the accuracy of the taxonomic identities produced by this OFRG experiment, we determined the nucleotide sequences for 117 clones distributed throughout the tree. For all but two of these clones, the taxonomic identities generated by this OFRG experiment were consistent with those generated by a nucleotide sequence analysis. Eighty-eight percent of the clones were affiliated with Ascomycota, while 12% belonged to BASIDIOMYCOTA: A large fraction of the clones were affiliated with the genera Fusarium (404 clones) and Raciborskiomyces (176 clones). Smaller assemblages of clones had high sequence identities to the Alternaria, Ascobolus, Chaetomium, Cryptococcus, and Rhizoctonia clades.

DNA Fingerprinting↗

Fungal community analysis by large-scale sequencing of environmental samples.

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.

Computational Biology↗

Abundant and diverse fungal microbiota in the murine intestine.

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.

Animals↗

Introns and splicing elements of five diverse fungi.

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.

Aspergillus nidulans↗