Open Access
Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).
Open Access
Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Copper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.
Open Access
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goikoetxea, María Arantzazu; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Cellulose is the most abundant biological polymer on Earth. Its chemical composition consists of D-glucose units linked by β-1,4- glycosidic bonds forming linear polymeric chains with a reducing and a non-reducing end. Cellulose chains may either adhere to each other, via hydrophobic and van der Waals interactions, forming crystalline structures or remain more loosely packaged (amorphous cellulose). Consequently, the physical structure and morphology of native cellulose is complex and not uniform. Biological degradation of cellulose depends on the action of three types of enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous regions of the cellulose fibbers generating new reducing and non-reducing ends, cellobiohydrolases attack the molecule ends yielding cellobiose units, and β-glucosidases hydrolyse cellobiose molecules yielding glucose. Cellobiohydrolases can be classified into two groups: type I (CBHI) and type II (CBHII), each having opposite chain-end specificities. CBHI prefer the reducing ends while CBHII act at non-reducing ends. By the screening of a genomic library from the basidiomycete Pleurotus ostreatus var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3, cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this enzymatic activity. Using this sequences as probe, it has been possible to know the conditions in which are expressed those genes. This has allowed the synthesis of the each gene cDNA and, by comparison of this sequence with the corresponding genomic sequence, the characterization of their structure. On the other hand, using the RFLP technique and a progeny of 80 monokaryons derived from the dikaryon N001, the five genes have been mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to the chromosome IV and the others to the chromosome VI.
Open Access
Selection of Pleurotus ostreatus strains in a genetic breeding program
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The basidiomycete Pleurotus ostreatus, commonly known as oyster mushroom, is the second largest edible mushroom crop behind the white button mushroom, Agaricus bisporus. It accounts for nearly one-quarter of the total worldwide mushroom production. Furthermore, P. ostreatus has a high industrial interest because it is a good source of enzymes and other products with biotechnological, industrial and medical applications, it is easy to cultivate and because of its good organoleptic characteristics. Since of 2003, our group research has carried out genetic breeding programs based on the determination of QTLs controlling production and quality in industrial cultures of this fungus. In this breeding program the first test consisted in putting under fructification conditions 130 strains obtained from the crossing of protoclon PC21 (P. ostreatus var. ostreatus wild strain) by a collection of monokarions derived from N001 (P. ostreatus var. florida commercial strain). For this purpose, 2 kg (3 repetitions per strain) bags of industrial sustrate were inoculated and cultivated at 21ºC. Mature fruiting bodies were collected and weighted daily during the fructification period. The second test was made using the six strains that performed the better in Test1, but were cultivated at 18ºC and with 15 repetitions per strain were performed. From this test, three strains were selected and used in Test3. In this test, other three strains obtained from the crossing between monokarions descending of N001 and selectioned for their high growth rate were introduced. In this test the weight of the bags was increased to 5 kg and the cultures were cultivated at 18ºC. The strains obtained from PC21 have good charactericts for mushroom size, with similar behaviour for yield and precocity. The strains obtained from the crosses between N001 descendants have better mushroom size and similar yield and precocity than N001, then breeding was obtained. The candidate strains for next tests are PC21xMA046 and PC21xMA027 for their high yield and the mushroom good features.
Open Access
Mapping of genomic regions (quantitative trait loci) controlling production and quality in industrial cultures of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2003) Larraya Reta, Luis María; Alfonso Esquíroz, Mikel; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua
Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.
Open Access
Diffusional properties of methanogenic granular sludge: 1H NMR characterization
(American Society for Microbiology, 2003) Lens, Piet N. L.; Gastesi Barasoain, Rakel; Vergeldt, Frank; Van Aelst, Adriaan C.; Pisabarro de Lucas, Gerardo; Van As, Henk; Producción Agraria; Nekazaritza Ekoizpena
The diffusive properties of anaerobic methanogenic and sulfidogenic aggregates present in wastewater treatment bioreactors were studied using diffusion analysis by relaxation time-separated pulsed-field gradient nuclear magnetic resonance (NMR) spectroscopy and NMR imaging. NMR spectroscopy measurements were performed at 22°C with 10 ml of granular sludge at a magnetic field strength of 0.5 T (20 MHz resonance frequency for protons). Self-diffusion coefficients of H2O in the investigated series of mesophilic aggregates were found to be 51 to 78% lower than the self-diffusion coefficient of free water. Interestingly, self-diffusion coefficients of H2O were independent of the aggregate size for the size fractions investigated. Diffusional transport occurred faster in aggregates growing under nutrient-rich conditions (e.g., the bottom of a reactor) or at high (55°C) temperatures than in aggregates cultivated in nutrient-poor conditions or at low (10°C) temperatures. Exposure of aggregates to 2.5% glutaraldehyde or heat (70 or 90°C for 30 min) modified the diffusional transport up to 20%. In contrast, deactivation of aggregates by HgCl2 did not affect the H2O self-diffusion coefficient in aggregates. Analysis of NMR images of a single aggregate shows that methanogenic aggregates possess a spin-spin relaxation time and self-diffusion coefficient distribution, which are due to both physical (porosity) and chemical (metal sulfide precipitates) factors.
Open Access
Computational prediction of protein-coding gene and annotation of DNA sequences with agronomic interest in Pleurotus ostreatus (Oyster Mushroom)
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Palma Dovis, Leopoldo; Peñas Parrila, María Manuela; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus, commonly known as oyster mushroom, is a commercially important edible fungus with interesting biotechnological properties. Quantitative trait loci (QTL) analyses are rare in fungi and little is known about their number, position, and genetic structure. Previous studies of our group have allowed the construction of a genetic linkage map of P. ostreatus var. florida, which has provided the basis for performing an efficient QTL analysis. In fact, there is a region of the chromosome VII of P. ostreatus where the most QTLs related to the production and precocity characters have been mapped. These quantitative traits are presumably under the control of a polygenic genetic system and could be associated with some chromosomal regions. The hypothesis of this work is that there is a region in the chromosome VII of protoclon PC15 (monokaryotic parental of the N001 dikaryotic strain) where exist genes which are responsible for the QTLs mentioned above. In order to test this hypothesis, we are developing a molecular QTL analysis through the sequencing of a region with an approximated size of 320 Kbp in chromosome VII (protoclon PC15). For this purpose, a BAC genomic library was constructed and two BAC clones spanning the region of interest are being sequenced. To carry out an efficient computational prediction of protein-coding genes and its annotation on the partial sequences obtained up to date, we have used different Internet resources such as BLASTx, BLASTp, BLASTn, and FGENESH trained on some basidiomycetes genomic data like Phanerochaete chrysosporium and Cryptococcus neoformans (SoftBerry). To our knowledge, this is the firs molecular QTL analysis performed on this edible mushroom.
Open Access
Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2001) Larraya Reta, Luis María; Pérez Garrido, María Gumersinda; Iribarren, Iñaki; Blanco Vaca, Juan Antonio; Alfonso Esquíroz, Mikel; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBa and matBb) and to correlate the presence of specific matBa and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.
Open Access
Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion
(National Academy of Sciences, 2009) Martínez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Pisabarro de Lucas, Gerardo; Lavín Trueba, José Luis; Oguiza Tomé, José Antonio; Producción Agraria; Nekazaritza Ekoizpena
Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β -1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.
Open Access
Genetic linkage map of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2000) Larraya Reta, Luis María; Pérez Garrido, María Gumersinda; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.