The intraspecific variability of mitochondrial genes of Agaricus bisporus reveals an extensive group I intron mobility combined with low nucleotide substitution rates.

Intraspecific mitochondrial variability was studied in ten strains of A. bisporus var. bisporus, in a strain representative of A. bisporus var. eurotetrasporus and in a strain of the closely related species Agaricus devoniensis. In A. bisporus, the cox1 gene is the richest in group I introns harboring homing endonuclease genes (heg). This study led to identify group I introns as the main source of cox1 gene polymorphism. Among the studied introns, two groups were distinguished according to the heg they contained. One group harbored heg maintained putatively functional. The other group was composed of eroded heg sequences that appeared to evolve toward their elimination. Low nucleotide substitution rates were found in both types of intronic sequences. This feature was also shared by all types of studied mitochondrial sequences, not only intronic but also genic and intergenic ones, when compared with nuclear sequences. Hence, the intraspecific evolution of A. bisporus mitochondrial genome appears characterized by both an important mobility (presence/absence) of large group I introns and by low nt substitution rates. This stringent conservation of mitochondrial sequences, when compared with their nuclear counterparts, appears irrespective of their apparent functionality and contrasts to what is widely accepted in fungal sequence evolution. This strengthens the usefulness of mtDNA sequences to get clues on intraspecific evolution.

The first set of expressed sequence tags (EST) from the medicinal mushroom Agaricus subrufescens delivers resource for gene discovery and marker development.

Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high-added-value products and extended agronomical valorization. The development of A. subrufescens-related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4,989 and 5,125 sequences were obtained in SP and CL libraries, respectively. About 87% of the EST had significant similarity with Agaricus bisporus-predicted proteins, and 79% correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49% of the sequences. Some gene families potentially involved in bioactive compound biosynthesis could be identified. A total of 232 simple sequence repeats (SSRs) were identified, and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery and molecular marker development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.

Diversity of Mycoplasma hominis clinical isolates from Bordeaux, France, as assessed by multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Mycoplasma hominis is an opportunistic human mycoplasma species that can cause various urogenital infections and, less frequently, extragenital infections. The objective of this work was to study the genetic diversity of this species using a molecular typing method based on multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). RESULTS: The genome content of M. hominis PG21 was analysed for tandem repeats (TRs), and five of the 130 TRs identified were selected for use in an MLVA assay. The method was based on GeneScan analysis of VNTR loci using multiplex PCR with fluorescent dyes and resolution by capillary electrophoresis. This approach was used on a collection of 210 urogenital and extragenital French clinical isolates collected between 1987 and 2009. Forty MLVA types were found. The discriminatory index of our MLVA scheme was 0.924. Using this new typing tool, persistent infection was suggested for six patients and new infection for one patient. Furthermore, mother-to-child transmission was confirmed in the two cases studied. Application of MLVA to a wide range of M. hominis isolates revealed high genotypic diversity and no obvious link between the MLVA type and the isolate year of collection, the patient's age or sex, the anatomical origin of the isolates or resistance to antibiotics was found. CONCLUSIONS: Our MLVA scheme highlights the high genetic heterogeneity of the M. hominis species. It seems too discriminatory to be used for large epidemiological studies but has proven its usefulness for molecular studies at the individual level.

A molecular contribution to the assessment of the Tricholoma equestre species complex.

In recent years, interest in the Tricholoma equestre species complex has increased because of several cases of severe and sometimes fatal rhabdomyolysis reported in France and Poland. These occurred after repeated consumption of large portions of T. equestre sporophores during consecutive meals, despite the fact that this species is renowned as a tasty edible wild mushroom. The T. equestre species complex includes three ectomycorrhizal species Tricholoma flavovirens (Pers.) S. Lundell, Tricholoma auratum (Paulet) Gillet, and T. equestre (L.) P. Kummer. All these species produce sporophores with intense yellow gills but are difficult to distinguish by morphological analyses at both the macroscopic and microscopic levels. In T. equestre, two additional varieties are recognized: T. equestre var. populinum (Christensen & Noordeloos) associated with Populus sp. and/or Betula sp. trees and sometimes recognized as Tricholoma frondosae (Kalamees & Shchukin) and T. equestre var. pallidifolia characterized by pale to white gills, frequently recognized as Tricholoma joachimii (Bon & Riva). To explore the taxonomic (species delimitation), ecological, and geographical extent and limits of the T. equestre species complex, we have carried out a molecular comparison of worldwide strains belonging to this complex by using sequences of two molecular markers: the internal transcript spacer (ITS)1/5.8S/ITS2 region of the nuclear ribosomal unit and the 5' part of the mitochondrial cox1 gene. Phylogenetic analyses support the placement of European T. equestre, T. flavovirens, and T. auratum strains as representatives of a single species. This species appears associated with various conifers trees, depending on the geographic origin (Pinus pinaster for T. auratum, Pinus sylvestris or Abies alba for T. equestre and T. flavovirens). However, in the context of a single T. equestre species, the geographical location could lead to the characterization of sub-species or varieties, as suggested by the gathering of the four Asian (Japanese) T. auratum strains in a strongly supported distinct phylogenetic clade. Moreover, our analysis strongly argues for considering T. joachimii and the synonymised T. equestre var. pallidifolia as two representatives of a different species not belonging to the T. equestre group. This species would be phylogenetically related to the Tricholoma columbetta species with which they share white gills. Similarly, the phylogenetic analysis of the molecular data and the lack of gene flow between the strains associated with broad-leaved trees and those of the T. equestre complex, rather argues for two distinct species depending on the ecological niche: T. frondosae under broad-leaved trees and T. equestre under conifers.

The 135 kbp mitochondrial genome of Agaricus bisporus is the largest known eukaryotic reservoir of group I introns and plasmid-related sequences.

At 135,005 nt, the mitochondrial genome in Agaricus bisporus represents the largest fungal mitochondrial genome sequenced to date. Its large size is mainly due to the presence of mobile genetic elements, including a total of 43 group I introns, three group II introns, and five DNA fragments that show sequence similarity to linear invertron-like plasmids. The introns are distributed in eight of the 15 protein coding genes. These introns contain a total of 61,092 nt (∼45.3% of the whole mitochondrial genome) and include representatives of most of the group I introns so far found in mitochondrial genomes of Basidiomycota. The plasmid-like sequences include 6730 nt total representing 5.0% of the genome. These sequences showed high-level similarities to two different mitochondrial plasmids reported for basidiomycete mushrooms: the autonomously replicating pEM in Agaricus bitorquis and the integrated linear plasmid sequences in Agrocybe aegerita and Moniliophthora perniciosa. Moreover, the plasmid-related sequences are located within or adjacent to two large (4559 nt) inverted repeats containing also two sets of mitochondrial tRNA genes. Our analyses are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution of the A. bisporus mitochondrial genome.

Agaricus subrufescens: A review.

Medicinal mushrooms have currently become a hot issue due to their various therapeutic properties. Of these, Agaricus subrufescens, also known as the "almond mushroom", has long been valued by many societies (i.e., Brazil, China, France, and USA). Since its discovery in 1893, this mushroom has been cultivated throughout the world, especially in Brazil where several strains of A. subrufescens have been developed and used as health food and alternative medicine. This article presents up-to-date information on this mushroom including its taxonomy and health promoting benefits. Medicinal properties of A. subrufescens are emphasized in several studies which are reviewed here. In addition, safety issues concerning the use of this fungus will be discussed.

Potential role of Mycoplasma hominis in interleukin (IL)-17-producing CD4+ T-cell generation via induction of IL-23 secretion by human dendritic cells.

BACKGROUND: Mycoplasma hominis, a human urogenital pathogen, is involved in genital and extragenital infections and arthritis, particularly in immunocompromised patients. The interleukin (IL) 23/T helper (Th) 17 axis is associated with inflammatory and autoimmune diseases. The aim of this study was to assess the IL-23 response to M. hominis in human dendritic cells (DCs) and the CD4(+) T-cell differentiation in response to M. hominis-infected DCs. METHODS: Human monocyte-derived DCs were cultured with phosphate-buffered saline, lipopolysaccharide, or M. hominis PG21. Cocultures with heterologous T cells were performed. Extracts from M. hominis were separated and incubated with DCs. Isolates from different clinical syndromes were tested. RESULTS: M. hominis induced the maturation of human DCs with predominant IL-23 secretion in a Toll-like receptor 2-dependent manner. The in vitro immunomodulatory capacity of M. hominis was contained in a lipoprotein-enriched fraction from the mycoplasma. M. hominis-activated DCs induced IL-17-producing CD4(+) T cells. Interestingly, clinical isolates differed in their ability to promote IL-23 secretion by DCs. CONCLUSIONS: Taken together, our findings demonstrate a major role for the IL-23/Th17 axis in the defense against M. hominis and indicate a potential role for these bacteria in inflammatory and autoimmune diseases.

The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns.

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a "Homing Endonuclease Gene" (heg) encoding a DNA endonuclease acting in transfer and site-specific integration ("homing") and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain) is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt) and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.

Performance of the COX1 gene as a marker for the study of metabolically active Pezizomycotina and Agaricomycetes fungal communities from the analysis of soil RNA.

In temperate forest soils, filamentous ectomycorrhizal and saprotrophic fungi affiliated to the Agaricomycetes and Pezizomycotina contribute to key biological processes. The diversity of soil fungal communities is usually estimated by studying molecular markers such as nuclear ribosomal gene regions amplified from soil-extracted DNA. However, this approach only reveals the presence of the corresponding genomic DNA in the soil sample and may not reflect the diversity of the metabolically active species. To circumvent this problem, we investigated the performance of the mitochondrial cytochrome c oxidase 1 (COX1)-encoding gene as a fungal molecular marker for environmental RNA-based studies. We designed PCR primers to specifically amplify Agaricomycetes and Pezizomycotina COX1 partial sequences and amplified them from both soil DNA and reverse-transcribed soil RNA. As a control, we also amplified the nuclear internal transcribed spacer ribosomal region from soil DNA. Fungal COX1 sequences were readily amplified from soil-extracted nucleic acids and were not significantly contaminated by nontarget sequences. We show that the relative abundance of fungal taxonomic groups differed between the different sequence data sets, with for example ascomycete COX1 sequences being more abundant among sequences amplified from soil DNA than from soil cDNAs.

Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species.

The cob/tRNA(Tyr) mitochondrial regions of Agrocybe aegerita and of the related species Agrocybe chaxingu display an unusual clustering of four microsatellite loci constituted by motifs of one to six nucleotides whose number of repeats varied from three to 18. In A. chaxingu, these microsatellite loci are followed by a small region bearing one additional microsatelite and one minisatellite locus constituted by an octanucleotide motif repeated 13-18 times. In A. aegerita, this latter region is deleted. This is the first evidence of such an accumulation of microsatellites in mitochondrial genomes. The analyses of the microsatellite loci in 11 A. aegerita and in four A. chaxingu wild strains have shown extensive intraspecific and interspecific variations in the number of tandem repeats (VNTRs), suggesting that these loci could represent powerful molecular markers for strain fingerprinting. Up to 23 different alleles were present in the 15 Agrocybe studied strains, allowing the definition of 12 different haplotypes.

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA.

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi.

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.