Molecular breeding of a novel <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis> strain possessing a heterologous laccase gene, <Emphasis Type="Italic">lccK</Emphasis>, driven by a constitutive promoter

Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds.     

Temperature sensitive mutant of Trichoderma reesei defective in secretion of cellulase

Summary Temperature sensitive mutants of Trichoderma reesei derived from hypersecretory strain RL-P37 were isolated and characterized. Compared to the parent strain, one mutant (LU-ts 1) grew well in the mycelial phase at both permissive (25°C) and non-permissive (37°C) temperatures. However, the secretion of overall protein and active cellulases was significantly reduced in the mutant at the higher temperature. No accumulation of active cellulases or intracellular proteins was observed in the mycelia of LU-ts 1 at 37°C. The inhibitory effects of temperature on cellulase secretion in LU-ts 1 were reversible. Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses confirmed that the secretion of the major cellulases was greatly reduced in LU-ts 1 at 37°C. Molecular characterization of the various temperature sensitive secretion mutants of T. reesei should help elucidate the crucial aspects of the secretory pathway of this cellulolytic fungus.     

2‐DE proteomic approach to the Botrytis cinerea secretome induced with different carbon sources and plant‐based elicitors

Botrytis cinerea is a phytopathogenic fungus infecting a number of crops (tomatoes, grapes and strawberries), which has been adopted as a model system in molecular phytopathology. B. cinerea uses a wide variety of infection strategies, which are mediated by a set of genes/proteins called pathogenicity/virulence factors. Many of these factors have been described as secreted proteins, and thus the study of this sub‐proteome, the secretome, under changing circumstances can help us to understand the roles of these factors, possibly revealing new loci for the fight against the pathogen. A 2‐DE, MALDI TOF/TOF‐based approach has been developed to establish the proteins secreted to culture media supplemented with different carbon sources and plant‐based elicitors (in this study: glucose, cellulose, starch, pectin and tomato cell walls). Secreted proteins were obtained from the culture media by deoxycholate‐trichloroacetic acid/phenol extraction, and 76 spots were identified, yielding 95 positive hits that correspond to 56 unique proteins, including several known virulence factors (i.e. pectin methyl esterases, xylanases and proteases). The observed increases in secretion of proteins with established virulence‐related functions indicate that this in vitro‐induction/proteome‐mining approach is a promising strategy for discovering new pathogenicity factors and dissecting infection mechanisms in a discrete fashion.     

Differential expression of cellulases and xylanases by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> grown on different carbon sources

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Summary Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel R _f values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

The infection cushion of Botrytis cinerea: a fungal ‘weapon’ of plant-biomass destruction

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall–degrading enzymes and plant cell death–inducing proteins. Parallel upregulation of sugar transport and sugar catabolism–encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.     

Production of extracellular proteins,cellulases and antifungal metabolites by Chaetomium globosum Kunze ex. Fr.

Chaetomium globosum has been well-known potential antagonist of several seed and soilborne fungus. Eight isolates of C. globosum were obtained from different sources and were identified by morphological characters. C. globosum isolates examined for the presence of extra cellular proteins, cellulases and antifungal metabolites in culture filtrate by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), thin-layer chromatography and high-performance liquid chromatography. Variation in the mycelial protein of C. globosum isolates was noted in the SDS-PAGE analysis. Different C. globosum isolates that showed more number of bands in protein profile was further screened for the production of cellulases in culture filtrate. Cellulase activity of C. globosum isolates revealed that maximum activity was observed in the isolate Cg-6 after 11?days of incubation, while Cg-2 had least activity. C. globosum isolates were tested for antibiotic production, among which three isolates viz. Cg-6, Cg-7 and Cg-5 were found to produce the antibiotic Chaetoglobosin A in the culture filtrate. The antibiotic Chaetoglobosin A appeared blue colour under UV spectrum with a wavelength of 250?nm.     

Genome-Wide Identification,Evolution, and Expression Profile of Aquaporin Genes in Coffea canephora in Response to Water Deficit

Aquaporins (APQs) belong to the major intrinsic protein superfamily and play a key role in the transport of water and other solutes across cell membranes. Coffea canephora is an evergreen shrub used for making instant coffees. Genome analysis of C. canephora identified 33 putative aquaporin genes assigned to five subfamilies including seven plasma membrane intrinsic proteins (PIP), 9 tonoplast intrinsic proteins (TIP), 11 NOD26-like intrinsic proteins (NIP), 3 small basic intrinsic proteins (SIP), and 3 X intrinsic proteins (XIP). Generally, the AQPs gene structure was conserved within each subfamily, with exon numbers ranging from one to five. The prediction of the aromatic/arginine selectivity filter (ar/R) and Froger’s positions indicated a noticeable difference in substrate specificity between subfamilies. Synteny analysis revealed high conservation of aquaporin genes in coffee. In silico expression analysis of the CcAQPs genes indicated that they were differentially expressed in various tissues. Members of CcPIPs and CcTIPs subfamilies were validated by real-time quantitative analysis in leaves of two genotypes of C. canephora with contrasting responses to water deficit (clone 14: drought-tolerant and clone 109A: drought-susceptible). Under severe water deficit, the relative expression of isoforms of both genes decreased in clone 14 compared with that under the irrigated condition, while clone 109A showed comparatively higher mRNA levels, with the exception of CcPIP1;2 in the stress condition. This study was the first to characterize and validate aquaporin genes in C. canephora in response to water deficit, and the findings may provide insights for biotechnological approaches to increase tolerance to drought.

     

Mapping the twin‐arginine protein translocation network of Bacillus subtilis

Bacteria employ twin‐arginine translocation (Tat) pathways for the transport of folded proteins to extracytoplasmic destinations. In recent years, most studies on bacterial Tat pathways addressed the membrane‐bound TatA(B)C subunits of the Tat translocase, and the specific interactions between this translocase and its substrate proteins. In contrast, relatively few studies investigated possible coactors in the TatA(B)C‐dependent protein translocation process. The present studies were aimed at identifying interaction partners of the Tat pathway of Bacillus subtilis, which is a paradigm for studies on protein secretion by Gram‐positive bacteria. Specifically, 36 interaction partners of the TatA and TatC subunits were identified by rigorous application of the yeast two‐hybrid (Y2H) approach. Our Y2H analyses revealed that the three TatA isoforms of B. subtilis can form homo‐ and heterodimers. Subsequently, the secretion of the Tat substrates YwbN and PhoD was tested in mutant strains lacking genes for the TatAC interaction partners identified in our genome‐wide Y2H screens. Our results show that the cell wall‐bound protease WprA is important for YwbN secretion, and that the HemAT and CsbC proteins are required for PhoD secretion under phosphate starvation conditions. Taken together, our findings imply that the Bacillus Tat pathway is embedded in an intricate protein–protein interaction network.     

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of glycosylation inhibitors: measurement of pH and temperature optima,protease and ion sensitivities

Summary The effects of protein glycosylation inhibitors were studied in Neocallimastix frontalis EB188. Low concentrations of tunicamycin and 2-deoxy-D-glucose inhibited zoospore germination, rhizoidal elongation, carbon source utilization and the production and secretion of cellulases and proteins. The carbohydrate-trimming inhibitors, deoxynojirimycin and glucono--lactone, had no measurable effect on rhizoidal growth and carbon source utilization. Cellulases (intracellular or extracellular) synthesized in the presence of glycosylation inhibitors were sensitive to -endoglycosidase H digestion, periodate modification, certain salts, changes in incubation temperature and pH, and protease. Anthrone staining of extracellular proteins confirmed the presence of glycoproteins. In N. frontalis EB188, glycosylation of protein and cellulase occurred and was important for cellular development and the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Comparison of <Emphasis Type="Italic">Chloroflexus aurantiacus</Emphasis> strain J-10-fl proteomes of cells grown chemoheterotrophically and photoheterotrophically

Chloroflexus aurantiacus J-10-fl is a thermophilic green bacterium, a filamentous anoxygenic phototroph, and the model organism of the phylum Chloroflexi. We applied high-throughput, liquid chromatography–mass spectrometry in a global quantitative proteomics investigation of C. aurantiacus cells grown under oxic (chemoorganoheterotrophically) and anoxic (photoorganoheterotrophically) redox states. Our global analysis identified 13,524 high-confidence peptides that matched to 1,286 annotated proteins, 242 of which were either uniquely identified or significantly increased in abundance under photoheterotrophic culture condition. Fifty-four of the 242 proteins are previously characterized photosynthesis-related proteins, including chlorosome proteins, proteins involved in the bacteriochlorophyll biosynthesis, 3-hydroxypropionate (3-OHP) CO₂ fixation pathway, and components of electron transport chains. The remaining 188 proteins have not previously been reported. Of these, five proteins were found to be encoded by genes from a novel operon and observed only in photoheterotrophically grown cells. These proteins candidates may prove useful in further deciphering the phototrophic physiology of C. aurantiacus and other filamentous anoxygenic phototrophs.     

Statistical optimization of cellulases production by <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis> QML-2 under solid-state fermentation and primary application to chitosan hydrolysis

Solid-state fermentation conditions for cellulases production by a newly isolated Penicillium chrysogenum QML-2 were investigated using statistical methods. At first, significant variables for cellulases production including (NH₄)₂SO₄, initial pH and inoculum size were screened by using Plackett-Burman Design. Then the optimal regions of the significant variables were investigated by using the method of steepest ascent. Finally, central composite design and response surface analysis were adopted to determine the optimal values of the significant variables and investigate the combined effects of each variable’s pair on cellulases production. The results showed that the optimal ranges of (NH₄)₂SO₄ concentration, initial pH and inoculum size for three types of cellulases activities were 1.97–2.15 g, pH 4.32–4.41 and 13.3–13.7% (v/w), respectively. Using the mixture of corn stover powder and wheat bran (CSP/WB, 1/1) as carbon source, the optimization resulted in 370.15, 101.76 and 321.56 U/g for maximal endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. Compared with maximum values of cellulases activities (endoglucanase activity 85.21 U/g, filter paper activity 16.62 U/g and β-glucosidase activity 67.68 U/g) obtained under unoptimized conditions, the optimization resulted in 3.34, 5.12 and 3.75 folds improvement for endoglucanase activity, filter paper activity and β-glucosidase activity, respectively. For chitosan hydrolysis, the crude cellulases had the optimal temperature of 55°C, pH of 4.4 and exhibited Michaelis constant (K _m) value of 8.34 mg/ml and maximum velocity (V _max) of 2.21 μmol glucosamine/min by 1 ml of the crude cellulases.