Coprinopsis cinerea intracellular lactonases hydrolyze quorum sensing molecules of Gram-negative bacteria.

Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-ß-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle.

Rhizomucor variabilis var. regularior and Hormographiella aspergillata infections in a leukemic bone marrow transplant recipient with refractory neutropenia.

Rhizomucor variabilis and Hormographiella aspergillata rarely cause human infections. This report details a fatal case of a 14-year-old female with leukemia posthematopoietic cell transplant and relapse with refractory pancytopenia. The patient first developed an R. variabilis var. regularior palate infection and later developed a cutaneous H. aspergillata infection while on posaconazole and caspofungin therapy.

Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes.

Gene silencing in fungi is often induced by dsRNA hairpin forming constructs the preparation of which can require multiple cloning steps. To simplify gene silencing in the filamentous fungi we have evaluated a high throughput cloning method for target sequences using the homobasidiomycete Coprinopsis cinerea, the GFP reporter and a commercially available vector system. The pSUPER RNAi System, which was developed for mammalian experiments, exploits the human H1 Polymerase III (Pol III) RNA gene promoter and expedites cloning/expression of specific user-defined oligonucleotide sequences to form short self-complimentary hairpins. Transformation of C. cinerea with pSUPER constructs harboring specific oligonucleotides (19 nt stem length) enabled recovery of transformants with reduced transcripts of the GFP transgene, that were less fluorescent in protein assays and microscopic phenotypes. This technological advance should expedite functional genomic studies in C. cinerea and has wider potential for utility in other homobasidiomycete and filamentous fungi.

Fatal pulmonary infection in a leukaemic patient caused by Hormographiella aspergillata.

A case of autopsy-proven fungal pneumonia in a relapsed leukaemia patient is reported. The fungus Hormographiella aspergillata was cultured from two bronchoalveolar fluid samples and identified through morphological examination and ITS2 sequence analysis. In addition, galactomannan was detected in eight consecutive serum samples, which suggested a co-infection with Aspergillus species. The patient was treated with caspofungin.

Purification, characterization and evaluation of extracellular peroxidase from two Coprinus species for aqueous phenol treatment.

Non-ligninolytic fungal peroxidases produced by Coprinus cinereus UAMH 4103 and Coprinus sp. UAMH 10067 were purified, characterized and evaluated as cost-effective alternatives to horseradish peroxidase for aqueous phenol treatment. Purified Coprinus peroxidases exhibited a molecular weight of 36 kDa on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although the catalytic properties of the two Coprinus peroxidases were nearly identical in both crude and purified forms, the stabilities were substantially different. The peroxidase from Coprinus sp. UAMH 10067 was more stable at 50 degrees C and under basic conditions (up to pH 10) than the enzyme from C. cinereus UAMH 4103. The former enzyme also performed better at pH 9 than the latter one in aqueous phenol treatment. The phenol removal efficiency of the Coprinus peroxidase was comparable to those of previously studied plant peroxidases. The broader working pH and higher thermal and alkaline stability of the peroxidase from Coprinus sp. UAMH 10067 may be advantageous for its application to industrial wastewater treatment.

Determination of biodiversity of Coprinus comatus using genotyping and metabolic profiling tools.

Coprinus comatus strains (CCMs) originating from Poland were identified using ITS region sequencing. Based on the sequences obtained, the genetic relationship between the CCM strains was determined and a clear separation of all strains into two main clusters was obtained. The Coprinus strains were also genetically characterized for the first time by the AFLP technique. The analysis showed that the CCMs separated into four main clusters and a high complication of a UPGMA-based dendrogram was achieved. C. comatus strains included in the analysis displayed an AFLP profile similarity level in the range from 44 to 66%. The highest similarity coefficient, 0.490, was found between CCM12 and CCM13, and the lowest (0.202) between the CCM2 and CCM5 isolates. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mycelial growth. The analysis allowed comparison of the functional diversity of the CCM strains and revealed a broad variability within the analyzed Coprinus species based on substrate utilization profiles. Significant differences (2-48) have been shown in the substrate richness values. The Biolog experiments proved to be a good profiling technology for studying the diversity in shaggy manes due to metabolic differences and demonstrated that all the strains might be considered individually. It is evident that the strain metabolic grouping does not correlate with the grouping based on the ITS sequences and AFLP profiles, however, some similarities may be observed.

A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

The coprophilous mushroom Coprinus radians secretes a haloperoxidase that catalyzes aromatic peroxygenation.

Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 microM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.

Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the large ribosomal subunit RNA: divergent domains, outgroups, and monophyly.

Phylogenetic relationships were investigated in the mushroom genus Coprinus based on sequence data from the nuclear encoded large-subunit rDNA gene. Forty-seven species of Coprinus and 19 additional species from the families Coprinaceae, Strophariaceae, Bolbitiaceae, Agaricaceae, Podaxaceae, and Montagneaceae were studied. A total of 1360 sites was sequenced across seven divergent domains and intervening sequences. A total of 302 phylogenetically informative characters was found. Ninety-eight percent of the average divergence between taxa was located within the divergent domains, with domains D2 and D8 being most divergent and domains D7 and D10 the least divergent. An empirical test of phylogenetic signal among divergent domains also showed that domains D2 and D3 had the lowest levels of homoplasy. Two equally most parsimonious trees were resolved using Wagner parsimony. A character-state weighted analysis produced 12 equally most parsimonious trees similar to those generated by Wagner parsimony. Phylogenetic analyses employing topological constraints suggest that none of the major taxonomic systems proposed for subgeneric classification is able to completely reflect phylogenetic relationships in Coprinus. A strict consensus integration of the two Wagner trees demonstrates the problematic nature of choosing outgroups within dark-spored mushrooms. The genus Coprinus is found to be polyphyletic and is separated into three distinct clades. Most Coprinus taxa belong to the first two clades, which together form a larger monophyletic group with Lacrymaria and Psathyrella in basal positions. A third clade contains members of Coprinus section Comati as well as the genus Leucocoprinus, Podaxis pistillaris, Montagnea arenaria, and Agaricus pocillator. This third clade is separated from the other species of Coprinus by members of the families Strophariaceae and Bolbitiaceae and the genus Panaeolus.