Putative hydrophobins of black poplar mushroom (Agrocybe cylindracea).

Hydrophobin proteins were extracted from Agrocybe cylindracea mycelia, the culture media (potato dextrose broth, PDB), and fruiting bodies. The putative hydrophobins obtained showed approximate sizes ranging from 8.0 to 25.0 kDa, dependent on their source. Multiple hydrophobin protein bands were detected in fruiting bodies. The hydrophobin yielded from aerial mycelia, or fruiting bodies, was approximately 6 mg/g dried weight. The crude extracts were examined for their properties in regards to surface modification, emulsification, and surface activity. Coating of hydrophobic Teflon sheet with crude extract made the surface significantly hydrophilic, whereas exposure of glass surfaces to extracts resulted in enhanced hydrophobicity. Crude extracts from culture media of A. cylindracea displayed emulsifying activity when mixed with hexane and could significantly reduce the surface tension of 60% ethanol and deionised water. The putative hydrophobin protein band from culture media (9.6 kDa), as analysed using LC-MS/MS, contained an amino acid fragment structurally similar to class I hydrophobin proteins from Basidiomycetes.

Biodegradation of polycyclic aromatic hydrocarbons by a thermotolerant white rot fungus Trametes polyzona RYNF13.

The biodegradation of three polycyclic aromatic hydrocarbons (PAHs), phenanthrene, fluorene, and pyrene, by a newly isolated thermotolerant white rot fungal strain RYNF13 from Thailand, was investigated. The strain RYNF13 was identified as Trametes polyzona, based on an analysis of its internal transcribed spacer sequence. The strain RYNF13 was superior to most white rot fungi. The fungus showed excellent removal of PAHs at a high concentration of 100 mg·L-1. Complete degradation of phenanthrene in a mineral salt glucose medium culture was observed within 18 days of incubation at 30°C, whereas 90% of fluorene and 52% of pyrene were degraded under the same conditions. At a high temperature of 42°C, the strain RYNF13 was still able to grow, and degraded approximately 68% of phenanthrene, whereas 48% of fluorene and 30% of pyrene were degraded within 32 days. Thus, the strain RYNF13 is a potential fungus for PAH bioremediation, especially in a tropical environment where the temperature can be higher than 40°C. The strain RYNF13 secreted three different ligninolytic enzymes, manganese peroxidase, laccase, and lignin peroxidase, during PAH biodegradation at 30°C. When the incubation temperature was increased from 30°C to 37°C and 42°C, only two ligninolytic enzymes, manganese peroxidase and laccase, were detectable during the biodegradation. Manganese peroxidase was the major enzyme produced by the fungus. In the culture containing phenanthrene, manganese peroxidase showed the highest enzymatic activity at 179 U·mL-1. T. polyzona RYNF13 was determined as a potential thermotolerant white rot fungus, and suitable for application in the treatment of PAH-containing contaminants.

Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

The transcription factor Rbf1 is the master regulator for b-mating type controlled pathogenic development in Ustilago maydis.

In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor.

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.