Fungal communities as dual indicators of river biodiversity and water quality assessment.

Given their ecological importance, bioindicators are used for the assessment of the health of river ecosystems. This study explored the fungal compositions and the potential of fungal taxa as bioindicators for indicating the water quality of the Mekong River, as the use of fungal indicators of the Mekong River was not previously well characterized. The Mekong River exhibited dynamic variations in both physicochemical/hydrochemical properties and fungal communities according to seasons and locations. The results revealed the dominance of alkaline earth metal ions and weak acids in the water. The magnesium-bicarbonate water type was found in the dry season, but the water became the chloride-calcium type or mixed type of magnesium-bicarbonate and chloride-calcium in the rainy season at downstream sites. Fungal composition analysis revealed the dominance of Chytridiomycota in the dry season and intermediate periods, and Ascomycota and Basidiomycota in the rainy season. The fungal communities were influenced by stochastic and deterministic assembly processes, mainly homogeneous selection, heterogeneous selection, and dispersal limitation. The extent of environmental filtering implied that some fungal taxa were affected by environmental conditions, suggesting the possibility of identifying certain fungal taxa suitable for being bioindicators of water quality. Subsequently, machine learning with recursive feature elimination identified specific fungal bins mostly consisting of Agaricomycetes (mainly Polyporales, Agaricales, and Auriculariales), Dothideomycetes (mainly Pleosporales), Saccharomycetes (mainly Saccharomycetales), Chytridiomycota, and Rozellomycota as bioindicators that could predict ambient and irrigation water quality with high selectivity and sensitivity. These results thus promote the use of fungal indicators to assess the health of the river.

Beneficial bacterial-Auricularia cornea interactions fostering growth enhancement identified from microbiota present in spent mushroom substrate.

Complex dynamic bacterial-fungal interactions play key roles during mushroom growth, ranging from mutualism to antagonism. These interactions convey a large influence on mushroom's mycelial and fruiting body formation during mushroom cultivation. In this study, high-throughput amplicon sequencing was conducted to investigate the structure of bacterial communities in spent mushroom substrates obtained from cultivation of two different groups of Auricularia cornea with (A) high yield and (B) low yield of fruiting body production. It was found that species richness and diversity of microbiota in group (A) samples were significantly higher than in group (B) samples. Among the identified 765 bacterial OTUs, 5 bacterial species found to exhibit high differential abundance between group (A) and group (B) were Pseudonocardia mangrovi, Luteimonas composti, Paracoccus pantotrophus, Sphingobium jiangsuense, and Microvirga massiliensis. The co-cultivation with selected bacterial strains showed that A. cornea TBRC 12900 co-cultivated with P. mangrovi TBRC-BCC 42794 promoted a high level of mycelial growth. Proteomics analysis was performed to elucidate the biological activities involved in the mutualistic association between A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794. After co-cultivation of A. cornea TBRC 12900 and P. mangrovi TBRC-BCC 42794, 1,616 proteins were detected including 578 proteins of A. cornea origin and 1,038 proteins of P. mangrovi origin. Functional analysis and PPI network construction revealed that the high level of mycelial growth in the co-culture condition most likely resulted from concerted actions of (a) carbohydrate-active enzymes including hydrolases, glycosyltransferases, and carbohydrate esterases important for carbohydrate metabolism and cell wall generation/remodeling, (b) peptidases including cysteine-, metallo-, and serine-peptidases, (c) transporters including the ABC-type transporter superfamily, the FAT transporter family, and the VGP family, and (d) proteins with proposed roles in formation of metabolites that can act as growth-promoting molecules or those normally contain antimicrobial activity (e.g., indoles, terpenes, ß-lactones, lanthipeptides, iturins, and ectoines). The findings will provide novel insights into bacterial-fungal interactions during mycelial growth and fruiting body formation. Our results can be utilized for the selection of growth-promoting bacteria to improve the cultivation process of A. cornea with a high production yield, thus conveying potentially high socio-economic impact to mushroom agriculture.

A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis.

Bacillus subtilis is commonly used as a host for heterologous protein production via plasmid-based expression system. In order to improve product safety, avoid carbon catabolite repression and lower production cost, a novel salt-inducible vector, pSaltExSePR5, was developed based on a natural plasmid of Lactobacillus plantarum BCC9546. Salt-inducible promoter opuAA and a DNA fragment encoding a signal peptide of subtilisin E (SubE) were sequentially added to the core shuttle vector to facilitate expression and secretion of a target protein in B. subtilis. To evaluate the effectiveness of this system under salt induction, a protease gene from Halobacillus sp. without its native signal sequence was inserted in the pSaltExSePR5 plasmid downstream of SubE signal sequence and transformed into B. subtilis WB800. Protease activities from cell-free supernatants of the recombinant bacteria cultures induced with 0.5-6% NaCl were analyzed. The highest protease activity of 9.1 U/ml was obtained after induction with 4% NaCl, while the non-induced culture exhibited activity of 0.128 U/ml. The results demonstrated that pSaltExSePR5 provides an alternative vector for efficient and simple production of heterologous proteins in B. subtilis with a safer and more economic inducer.

Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement.

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

Analysis of the interaction between human kidney anion exchanger 1 and kanadaptin using yeast two-hybrid systems

Kidney anion exchanger adaptor protein (Kanadaptin) is a protein which interacts with the cytoplasmic N-terminal domain of kidney anion exchanger 1 (kAE1) and was first detected in mice using the yeast two-hybrid system and was also found to co-localize with kAE1 in rabbit a-intercalated cells. Impaired trafficking of human kAE1 can result in the kidney disease-distal renal tubular acidosis (dRTA), and defective interaction between human kAE1 and kanadaptin may cause this trafficking impairment and be the basis for dRTA pathogenesis. However, it is unknown whether kAE1 can really interact with kanadaptin in humans. We have thus investigated the interaction between human kAE1 and human kanadaptin by using both Gal4 and LexA yeast two-hybrid systems. It was found that co-expression of Gal4DBD fused to the cytoplasmic N-terminal domain of kAE1 and Gal4AD fused to kanadaptin could not activate the transcription of the ADE2, HIS3 and lacZ reporters in the Gal4 system. A similar result was obtained for the interaction between B42AD fused to the cytoplasmic N-terminal domain of kAE1 and LexA fused to kanadaptin in activation of lacZ transcription in the LexA system. The absence of interaction between the fusion proteins in both yeast two-hybrid systems raises the possibility that kAE1 may not interact with kanadaptin in human cells. Considerably different structures of both kAE1 and kanadaptin in mice and humans may lead to different binding properties of the proteins in these two species.

Expression of biologically active crustacean hyperglycemic hormone (CHH) of Penaeus monodon in Pichia pastoris.

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the alpha-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 micro g/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Secretion of Pem-CMG, a peptide in the CHH/MIH/GIH family of Penaeus monodon, in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae.

The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.