Cloning and sequencing of phenol oxidase 1 (pox1) gene from Pleurotus ostreatus

The gene (pox1) encoding a phenol oxidase 1 from Pleurotus ostreatus was sequenced and the corresponding pox1-cDNA was also synthesized, cloned and sequenced. The isolated gene is flanked by an upstream region called the promoter (399 bp) prior to the start codon (ATG). The putative metalresponsive elements (MREs) were determined in the promoter region, where MRE 1, 2 and 3 were located in positions -20, -62 and -389, respectively. Functional TATA consensus sequences were recognized in positions -78 and -245, while CAAT consensus sequence was recognized in position -171. The putative GC boxes consensus sequences were recognized in positions -175 and -344, and xenobiotic-responsive elements (XREs) in positions -100 and -270. The pox1-DNA gene consists of 2656 bp, with the coding sequence being interrupted by 19 introns. The nucleotide sequence of cDNA (pox1- cDNA) was found to contain an ORF of 1590 bp capable of coding for a protein of 529 amino acid residues. The signal peptide was predicted to be 23 amino acids in length using SIGNALP 3.0 program. Northern blot analysis revealed that strong transcriptional induction was observed in the coppersupplemented cultures for pox1 gene.Key words: Pleurotus, cDNA, pox1, gene promoter, putative sequences, northern blot analysis, copper

Optimization of cellulase and &#223-glucosidase induction by sugarbeet pathogen Sclerotium rolfsii

The ability to produce cellulose degrading enzymes by sugarbeet pathogen Sclerotium rolfsii Sacc. in liquid synthetic media with carboxymethy cellulose (CMC) as inducer was studied. Several culturalconditions were examined to assess their effect in optimizing enzymes production. Shaking cultures gave higher yields of cellulases compared with static ones. Aspargine supplement was the best nitrogen source, especially at 3.0 g/l concentration, in promoting enzyme production. Variation of cellulose/xylan ratio in the culture medium showed that cellulose and xylan induced both cellulases synthesis but cellulose being the most effective specific substrate. The influence of different inhibitorson enzymes production by S. rolfsii was also studied. Cyclohexmide and ethidium bromide inhibited protein synthesis by S. rolfsii. Moreover, glucose repressed cellulase synthesis in S. rolfsii

Phylogenetic and Expression Studies of Small GTP-Binding Proteins in Solanum lycopersicum Super Strain B

This investigation involved a comparative analysis of the small GTPase superfamily in S. lycopersicum super strain B compared to their analogues in leguminous and other non-leguminous species. The small GTPases superfamily members were recognized by tBLASTn searches. The sequences of amino acid were aligned using Clustal Omega and the analysis of phylogeny was performed with the MEGA7 package. Protein alignments were applied for all studied species. Three-dimensional models of RABA2, ROP9, and ROP10 from Solanum lycopersicum “Super strain B” were performed. The levels of mRNA of the Rab, Arf, Rop, and Ran subfamilies were detected in aerial tissues vs. roots. Significant divergences were found in the number of members and groups comprising each subfamily of the small GTPases and Glycine max had the highest count. High expression of Rab and Arf proteins was shown in the roots of legumes whilst in non-legume plants, the highest values were recorded in aerial tissues. S. lycopersicum super strain B had the highest expression of Rab and Arf proteins in its aerial tissues, which may indicate that diazotroph strains have supreme activities in the aerial tissues of strain B and act as associated N-fixing bacteria. The phylogenies of the small GTPase superfamily of the studied plants did not reveal asymmetric evolution of the Ra, Arf, Rop, and Ran subfamilies. Multiple sequence alignments derived from each of the Rab, Arf, and Rop proteins of S. lycopersicum super strain B showed a low frequency of substitutions in their domains. GTPases superfamily members have definite functions during infection, delivery, and maintenance of N2-fixing diazotroph but show some alterations in their function among S. lycopersicum super strain B, and other species.</jats:p

One fungus, which genes?: development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1-D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial beta-tubulin II (TUB2); iv) gamma-actin (ACT); v) translation elongation factor 1-alpha (TEF1 alpha); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5-6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1 alpha. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1 alpha, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail

Metagenomic analysis of fungal taxa inhabiting Mecca region, Saudi Arabia

AbstractThe data presented contains the sequences of fungal Internal Transcribed Spacer (ITS) and 18S rRNA gene from a metagenome of the Mecca region, Saudi Arabia. Sequences were amplified using fungal specific primers, which amplified the amplicon aligned between the 18S and 28S rRNA genes. A total of 460 fungal species belonging to 133 genera, 58 families, 33 orders, 13 classes and 4 phyla were identified in four contrasting locations. The raw sequencing data used to perform this analysis along with FASTQ file are located in the NCBI Sequence Read Archive (SRA) under accession numbers: SRR3150823, SRR3144873, SRR3150825 and SRR3150846

Susceptibility and Diversity in the Therapy-Refractory Genus Scedosporium

Scedosporium species show decreased susceptibility to the majority of systemic antifungal drugs. Acquired resistance is likely to disseminate differentially with the mode of exchange of genetic material between lineages. Inter- and intraspecific diversities of Scedosporium species were analyzed for three partitions (rDNA internal transcribed spacer gene [ITS], partial β-tubulin gene, and amplified fragment length polymorphism profiles), with the aim to establish distribution of resistance between species, populations, and strains. Heterogeneity of and recombination between lineages were determined, and distances between clusters were calculated using a centroid approach. Clinical, geographic, and antifungal data were plotted on diversity networks. Scedosporium minutisporum, Scedosporium desertorum, and Scedosporium aurantiacum were distinguished unambiguously in all partitions and had differential antifungal susceptibility profiles (ASP). Pseudallescheria fusoidea and Pseudallescheria ellipsoidea were indistinguishable from Scedosporium boydii. Pseudallescheria angusta took an intermediate position between Scedosporium apiospermum and S. boydii. Scedosporium boydii and S. apiospermum had identical ASP. Differences in (multi)resistance were linked to individual strains. S. apiospermum and S. boydii showed limited interbreeding and were recognized as valid, sympatric species. The S. apiospermum/S. boydii group, comprising the main clinically relevant Scedosporium species, consists of separate lineages and is interpreted as a complex undergoing sympatric evolution with incomplete lineage sorting. In routine diagnostics, the lineages in S. apiospermum/S. boydii are indicated with the umbrella descriptor "S. apiospermum complex"; individual species can be identified with rDNA ITS with 96.3% confidence. Voriconazole is recommended as the first-line treatment; resistance against this compound is rare