Bacterial effectors mimicking ubiquitin-proteasome pathway tweak plant immunity.

Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm. Mostly T3Es are secreted into the plant cells to establish pathogenicity by affecting the vital plant process viz. metabolic pathways, signal transduction and hormonal regulation. Ubiquitin-26S proteasome system (UPS) exists as one of the important pathways in plants to control plant immunity and various cellular processes by employing several enzymes and enzyme components. Pathogenic and non-pathogenic bacteria are found to secrete effectors into plants with structural and/or functional similarity to UPS pathway components like ubiquitin E3 ligases, F-box domains, cysteine proteases, inhibitor of host UPS or its components, etc. The bacterial effectors mimic UPS components and target plant resistance proteins for degradation by proteasomes, thereby taking control over the host cellular activities as a strategy to exert virulence. Thus, the bacterial effectors circumvent plant cellular pathways leading to infection and disease development. This review highlights known bacterial T3E and T4E proteins that function and interfere with the ubiquitination pathway to regulate the immune system of plants.

Bacillus aryabhattai TFG5-mediated synthesis of humic substances from coir pith wastes.

BACKGROUND: Humic substances (HS) form the largest proportion among all the constituents of soil organic matter and are a key component of the terrestrial ecosystem. HS plays a multifunctional role in the environment by controlling the biogeochemical carbon cycle, providing nutrients and bio-stimulants for plant growth, and interacting with inorganic and organic pollutants. The rate of formation of HS in soils determines its productivity and carbon sequestration capacity. Enhancement of HS synthesis in the soil through the microbial route not only increases CO2 sequestration but also mitigates the greenhouse gas emissions in the environment. RESULT: In this study, we attempted to understand the mechanism of formation and enhancement of HS from coir pith wastes using the tyrosinase produced by Bacillus aryabhattai TFG5. The bacterium TFG5 isolated from the termite garden produced the tyrosinase (1.34 U mL-1) and laccase (2.1 U mL-1) at 48 h and 60 h of fermentation, respectively. The extracellular tyrosinase from B. aryabhattai TFG5 was designated as TyrB. Homology modeling of TyrB revealed a structure with a predicted molecular mass of 35.23 kDa and two copper ions in the active center with its conserved residues required for the tyrosinase activity. TyrB efficiently transformed and polymerized standard phenols, such as p-cresol, p-hydroxyl benzoic acid, Levo DOPA, and 2,6 DMP, besides transforming free phenols in coir pith wash water (CWW). Additionally, UV-Vis and FT-IR spectra of the degradation products of the coir pith treated with TyrB revealed the formation of HS within 3 days of incubation. Furthermore, the E472/664 ratio of the degradation products revealed a higher degree of condensation of the aromatic carbons and the presence of more aliphatic structures in the HS. CONCLUSION: The results confirmed the influence of TyrB for the effective synthesis of HS from coir pith wastes. The results of the present study also confirm the recently accepted theory of humification proposed by the International Humic Substances Society.

Saccharification of sunflower stalks using lignocellulases from a fungal consortium comprising Pholiota adiposa and Armillaria gemina.

Lignocellulases from Armillaria gemina and Pholiota adiposa are efficient in hydrolyzing aspen and poplar biomass, respectively. In the present study, lignocellulosic enzymes obtained from a fungal consortium comprising P. adiposa and A. gemina were used for the saccharification of sunflower stalks. Sunflower stalks were thermochemically pretreated using 2 % NaOH at 50 °C for 24 h. The saccharification process parameters including substrate concentration, enzyme loading, pH, and temperature were optimized using response surface methodology to improve the saccharification yield. The highest enzymatic hydrolysis (84.3 %) was obtained using the following conditions: enzyme loading 10 FPU/g-substrate, substrate 5.5 %, temperature 50 °C, and pH 4.5. The hydrolysis yield obtained using the enzymes from the fungal consortium was equivalent to that obtained using a mixture of commercial enzymes Celluclast and Novozyme ß-glucosidase. Addition of up to 500 ppm of heavy metal ions (As, Cu, Fe, Mn, Ni, Pb, and Zn) during saccharification did not significantly affect the saccharification yield. Thus, the biomass grown for phytoremediation of heavy metals can be used for the production of reducing sugars followed by ethanol fermentation.

Characterization of a ß-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment.

A highly efficient ß-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa ß-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa ß-1,4-mannanase showed the highest activity toward locust bean gum (V max = 1,990 U/mg protein, K m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa ß-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway(®) (113 mg/g-substrate), P. adiposa ß-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa ß-1,4-mannanase a good candidate as a new commercial ß-1,4-mannanase to improve biomass pretreatment.

Optimization of ß-glucosidase production by a strain of Stereum hirsutum and its application in enzymatic saccharification.

A high beta-glucosidase (BGL)-producing strain, Stereum hirsutum, was identified and isolated and showed a maximum BGL activity (10.4 U/ml) when cultured with Avicel and tryptone as the carbon and nitrogen sources, respectively. In comparison with other BGLs, BGL obtained from S. hirsutum showed a higher level of activity to cellobiose (V(max) = 172 U/mg, and k(cat) = 281/s). Under the optimum conditions (600 rpm, 30°C, and pH 6.0), the maximum BGL activity of 10.4 U/ml with the overall productivity of 74.5 U/l/h was observed. BGL production was scaled up from a laboratory scale (7-L fermenter) to a pilot scale (70-L fermenter). When S. hirsutum was cultured in fed-batch culture with rice straw as the carbon source in a 70-L fermenter, a comparable productivity of 78.6 U/l/h was obtained. Furthermore, S. hirsutum showed high levels of activity of other lignocellulases (cellobiohydrolase, endoglucanase, xylanase, and laccase) that are involved in the saccharification of biomasses. Application of S. hirsutum lignocellulases in the hydrolysis of Pinus densiflora and Catalpa ovata showed saccharification yields of 49.7% and 43.0%, respectively, which were higher than the yield obtained using commercial enzymes.

Molecular cloning and characterization of a GH11 endoxylanase from Chaetomium globosum, and its use in enzymatic pretreatment of biomass.

An endo-1,4-ß-xylanase gene, xylcg, was cloned from Chaetomium globosum and successfully expressed in Escherichia coli. The complete gene of 675 bp was amplified, cloned into the pET 28(a) vector, and expressed. The optimal conditions for the highest activity of the purified recombinant XylCg were observed at a temperature of 40 °C and pH of 5.5. Using oat-spelt xylan, the determined K m, V max, and k cat/K m values were 0.243 mg ml⁻¹, 4,530 U mg⁻¹ protein, and 7,640 ml s⁻¹ mg⁻¹, respectively. A homology model and sequence analysis of XylCg, along with the biochemical properties, confirmed that XylCg belongs to the GH11 family. Rice straw pretreated with XylCg showed 30 % higher conversion yield than the rice straw pretreated with a commercial xylanase. Although xylanases have been characterized from fungal and bacterial sources, C. globosum XylCg is distinguished from other xylanases by its high catalytic efficiency and its effectiveness in the pretreatment of lignocellulosic biomass.

Characterization of a recombinant aryl ß-glucosidase from Neosartorya fischeri NRRL181.

An isolated gene from Neosartorya fischeri NRRL181 encoding a ß-glucosidase (BGL) was cloned, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,467 bp, capable of encoding a polypeptide of 488 amino acid residues. The gene was over-expressed in Escherichia coli, and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified recombinant BGL showed a high level of catalytic activity, with V (max) of 886 µmol min(-1) mg-protein(-1) and a K (m) of 68 mM for p-nitrophenyl-ß-D: -glucopyranoside (pNPG). The optimal temperature for enzyme activity was about 40°C, and the optimal pH was about 6.0. A homology model of N. fischeri BGL1 was constructed based on the X-ray crystal structure of Phanerochaete chrysosporium BGLA. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose shed light on the unique substrate specificity of N. fischeri BGL1 only towards pNPG.

Antitumor activity of methylan polysaccharide derivatives.

Methylan polysaccharide derivatives were prepared by dialkylaminoalkylation and reductive amination followed by quaternization. Their antitumor activity was investigated and a relationship between structure and activity is suggested. For quaternized DEAE-methylan at only 75 mug ml(-1), tumor cell proliferation was suppressed by 58-84% in three cell lines tested in the order Colo < Hela < HepG2.