Sergio Lobos

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and expression of a laccase gene from the ligninolytic basidiomycete Ceriporiopsis subvermispora

A gene encoding laccase has been isolated from a genomic library of the white-rot basidiomycete Ceriporiopsis subvermispora constructed in Lambda GEM-11. This gene (Cs-lcs1) contains an open reading frame of 2215 bp, encoding a mature protein of 499 amino acids with a 21-residue signal peptide. The protein sequence exhibits between 63 and 68% identity with laccases from other basidiomycetes and shares with all of them 10 conserved histidines and one cysteine involved in the coordination of copper atoms at the active site of the enzyme. The gene possesses 11 introns, with splicing junctions and internal lariat formation sites adhering to the GT-AG and CTRAY rules, respectively. The upstream region of Cs-lcs1 contains a TATA box, two CAAT sites, five putative metal response elements and a ACE1 element. In agreement with the presence of the latter element, transcription of Cs-lcs1 is activated by copper and silver, as shown by Northern blot and reverse transcription followed by DNA amplification analyses. Based on Southern blot analysis, Cs-lcs1 appears to be the only gene encoding laccase in C. subvermispora.

Characterization of three new manganese peroxidase genes from the ligninolytic basidiomycete Ceriporiopsis subvermispora.

Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese.

DNA Isolation and AFLP Fingerprinting of Nectarine and Peach Varieties (Prunus persica)

Traditional identification of peach and nectarine varieties relies on the assessment of agronomic traits of the adult plant. This leads to a significant delay of time, constraints to breeders in the surveillance of germplasm and a risk for fruit growers and exporters. We describe a method for rapid assessment of peach and nectarine varieties based on AFLP fingerprinting and extraction of high quality DNA. The best primer pairs were selected from 64 primer combinations that reliably distinguished 8 peach and 6 nectarine varieties. A graphical representation of the detected polymorphisms was shown to simplify the analysis.

Isoenzyme Multiplicity and Characterization of Recombinant Manganese Peroxidases from Ceriporiopsis subvermispora and Phanerochaete chrysosporium

ABSTRACT We expressed cDNAs coding for manganese peroxidases (MnPs) from the basidiomycetes Ceriporiopsis subvermispora (MnP1) andPhanerochaete chrysosporium (H4) under control of the α-amylase promoter from Aspergillus oryzae inAspergillus nidulans. The recombinant proteins (rMnP1 and rH4) were expressed at similar levels and had molecular masses, both before and after deglycosylation, that were the same as those described for the MnPs isolated from the corresponding parental strains. Isoelectric focusing (IEF) analysis of rH4 revealed several isoforms with pIs between 4.83 and 4.06, and one of these pIs coincided with the pI described for H4 isolated from P. chrysosporium (pI 4.6). IEF of rMnP1 resolved four isoenzymes with pIs between 3.45 and 3.15, and the pattern closely resembled the pattern observed with MnPs isolated from C. subvermispora grown in solid-state cultures. We compared the abilities of recombinant MnPs to use various substrates and found that rH4 could oxidize o-dianisidine and p-anisidine without externally added manganese, a property not previously reported for this MnP isoenzyme from P. chrysosporium.

Binding of nuclear proteins to the promoter region of the laccase gene Cs-lcs1 from the basidiomycete Ceriporiopsis subvermispora

Abstract The white rot basidiomycete Ceriporiopsis subvermispora secretes the ligninolytic enzymes manganese-dependent peroxidase (MnP) and laccase to the extracellular medium. The promoter region of the laccase gene ( Cs-lcs1 ) possesses several putative metal responsive elements (MRE), as well as a putative target site responding to copper termed ACE, similar to the one found in yeast. In this work, we show by electrophoretic mobility-shift assays that the migration of DNA probes containing either MRE sites or the ACE element are retarded in their mobility after incubation with nuclear extracts from C. subvermispora . Competition experiments suggested the presence of defined binding proteins recognizing these elements.

DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.

Molecular analysis of Broussonetia papyrifera (L.) Vent. (Magnoliophyta: Urticales) from the Pacific, based on ribosomal sequences of nuclear DNA

Abstract Broussonetia papyrifera (L.) Vent. (Magnoliophyta: Urticales), or paper mulberry, is a species of Asian origin dispersed by humans throughout the Pacific. Our aim is to evaluate the genetic variability of this plant in order to determine its potential as a commensal species for studying the mobility and/or migratory movements of the people that carried it. For this study, we analysed the non-coding transcribed spacer sequences (ITS) of ribosomal nuclear DNA found in samples of B. papyrifera collected in Remote Oceania and Taiwan. Our results show three genotypes: the Pacific samples form a distinct and homogenous subgroup, while the Taiwanese accessions present two genotypes. We discuss the relevance of these results in the context of the dispersal of B. papyrifera in the Pacific and its association with Austronesian migration history.

Ancient and modern introduction of Broussonetia papyrifera ([L.] Vent.; Moraceae) into the Pacific: genetic, geographical and historical evidence

Broussonetia papyrifera (L.) Vent. (Moraceae), or paper mulberry, is a species of cultural importance in South East Asia, East Asia and the Pacific. Originally from mainland South East Asia or East Asia, this plant was introduced into the Pacific range by prehistoric Austronesian voyagers. We used non-coding internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA and inter-simple sequence repeat (ISSR) on 79 samples of B. papyrifera from different islands of Remote Oceania, and South East Asia and East Asia. Our results show an absence of genetic diversity in the introduced range of Remote Oceania, with the sole exception of Hawaii. By contrast, Asian samples show genetic diversity. The data obtained suggest a prehistoric human-mediated introduction of this species from East Asia to Remote Oceania and a second, possibly historic, human-mediated introduction to Hawaii.