Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi.

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

Quantitative Proteome Profiling Reveals Cellobiose-Dependent Protein Processing and Export Pathways for the Lignocellulolytic Response in Neurospora crassa.

Filamentous fungi are intensively used for producing industrial enzymes, including lignocellulases. Employing insoluble cellulose to induce the production of lignocellulases causes some drawbacks, e.g., a complex fermentation operation, which can be overcome by using soluble inducers such as cellobiose. Here, a triple ß-glucosidase mutant of Neurospora crassa, which prevents rapid turnover of cellobiose and thus allows the disaccharide to induce lignocellulases, was applied to profile the proteome responses to cellobiose and cellulose (Avicel). Our results revealed a shared proteomic response to cellobiose and Avicel, whose elements included lignocellulases and cellulolytic product transporters. While the cellulolytic proteins showed a correlated increase in protein and mRNA levels, only a moderate correlation was observed on a proteomic scale between protein and mRNA levels (R2 = 0.31). Ribosome biogenesis and rRNA processing were significantly overrepresented in the protein set with increased protein but unchanged mRNA abundances in response to Avicel. Ribosome biogenesis, as well as protein processing and protein export, was also enriched in the protein set that showed increased abundance in response to cellobiose. NCU05895, a homolog of yeast CWH43, is potentially involved in transferring a glycosylphosphatidylinositol (GPI) anchor to nascent proteins. This protein showed increased abundance but no significant change in mRNA levels. Disruption of CWH43 resulted in a significant decrease in cellulase activities and secreted protein levels in cultures grown on Avicel, suggesting a positive regulatory role for CWH43 in cellulase production. The findings should have an impact on a systems engineering approach for strain improvement for the production of lignocellulases.IMPORTANCE Lignocellulases are important industrial enzymes for sustainable production of biofuels and bio-products. Insoluble cellulose has been commonly used to induce the production of lignocellulases in filamentous fungi, which causes a difficult fermentation operation and enzyme loss due to adsorption to cellulose. The disadvantages can be overcome by using soluble inducers, such as the disaccharide cellobiose. Quantitative proteome profiling of the model filamentous fungus Neurospora crassa revealed cellobiose-dependent pathways for cellulase production, including protein processing and export. A protein (CWH43) potentially involved in protein processing was found to be a positive regulator of lignocellulase production. The cellobiose-dependent mechanisms provide new opportunities to improve the production of lignocellulases in filamentous fungi.

Simultaneous determination of intracellular nucleotides and coenzymes in Yarrowia lipolytica producing lipid and lycopene by capillary zone electrophoresis.

Yarrowia lipolytica is an oleaginous yeast with promise in producing terpenoids such as lycopene. Though methods for analyzing primary metabolic intermediates have been established, further work is needed to better analyze nucleotides and coenzymes. Here, we presented an optimized method for the separation of nucleotides and coenzymes in Y. lipolytica using the capillary electrophoresis. The separation of twelve metabolites including four coenzymes, five nucleotides and three nucleosides was achieved within 32min using a voltage of 15kV and 70mM sodium carbonate/hydrogencarbonate buffer with 1.0% ß-CD at pH 10. The results show that the concentrations of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate changed significantly between lycopene producing strain and the control, indicating that these two metabolites may be closely related with lycopene production. The optimized method provides a useful approach for future metabolic analysis of fermentation process as well as industrial strain improvement.

Rhizobacter bergeniae sp. nov., isolated from the root of Bergenia scopulosa.

A yellowish-pigmented bacterium, designated strain PLGR-1(T), was isolated from the root of Bergenia scopulosa collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain PLGR-1(T) were Gram-stain-negative, strictly aerobic, rod-shaped, non-spore-forming and motile with a single polar flagellum. Growth occurred at 7-33 °C (optimum, 25-28 °C), at pH 5.0-10.0 (optimum, pH 6.0-7.0) and with 0-0.5 % (w/v) NaCl (optimum, 0 %). The predominant respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). The major polyamines were putrescine and 2-hydroxyputrescine and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was 69.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain PLGR-1(T) belonged to the class Betaproteobacteria and formed a tight phyletic lineage with members of the genus Rhizobacter. Strain PLGR-1(T) was most closely related to Rhizobacter dauci DSM 11587(T) and Rhizobacter fulvus DSM 19916(T), with 16S rRNA gene sequence similarities of 98.5 and 98.0 %, respectively. The DNA-DNA relatedness values between strain PLGR-1(T) and the type strains of Rhizobacter dauci and Rhizobacter fulvus were 46.3 and 14.7 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain PLGR-1(T) is considered to represent a novel species of the genus Rhizobacter, for which the name Rhizobacter bergeniae sp. nov. is proposed. The type strain is PLGR-1(T) ( = CCTCC AB 2013018(T) = KCTC 32299(T) = LMG 27607(T)).

Asticcacaulis endophyticus sp. nov., a prosthecate bacterium isolated from the root of Geum aleppicum.

A strictly aerobic, light-yellow-coloured, stalked bacterium, designated strain ZFGT-14(T), was isolated from the root of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi province, north-west China, and was subjected to a taxonomic study using a polyphasic approach. This novel isolate grew at 7-33 °C (optimum 25-28 °C) and pH 6.0-10.0 (optimum pH 7.0-8.0). Flexirubin-type pigments were not produced. Cells were Gram-stain-negative, rod-shaped and motile with a single polar flagellum. The predominant respiratory quinone was Q-10. The major cellular fatty acids were summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), C16 : 0, C19 : 0 cyclo ω8c and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and the major polar lipids were phosphatidylglycerol and glycolipids. The DNA G+C content was 57.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZFGT-14(T) was most closely related to the genus Asticcacaulis and had low sequence similarity (95.0-95.9 %) with all species with validly published names within the genus Asticcacaulis. Based on the phenotypic, phylogenetic and genotypic data, strain ZFGT-14(T) is considered to represent a novel species of the genus Asticcacaulis, for which the name Asticcacaulis endophyticus sp. nov. is proposed. The type strain is ZFGT-14(T) ( = CCTCC AB 2013012(T) = KCTC 32296(T) = LMG 27605(T)).

Pseudoxanthomonas gei sp. nov., a novel endophytic bacterium isolated from the stem of Geum aleppicum.

A Gram stain-negative, strictly aerobic, rod-shaped, non-motile and deep-yellow-coloured bacterial strain, designated ZFJR-3(T), was isolated from the stem of Geum aleppicum Jacq. collected from Taibai Mountain in Shaanxi Province, north-west China, and characterized by using a polyphasic approach. The novel isolate grew optimally at 25-28 °C and in the absence of NaCl. Flexirubin-type pigments were produced. The predominant respiratory quinone was ubiquinone-8 (Q-8) and the major cellular fatty acids were iso-C15:0 (29.2 %), iso-C16:0 (18.5 %), summed feature 9 (comprising iso-C17:1 ω9c and/or C16:0 10-methyl; 8.8 %), C16:1 ω7c alcohol (8.8 %), iso-C11:0 3-OH (6.9 %) and iso-C11:0 (6.8 %). The DNA G+C content was 66.1 mol %. The only polyamine was spermidine and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain ZFJR-3(T) belongs to the genus Pseudoxanthomonas and was most closely related to Pseudoxanthomonas yeongjuensis KCTC 22757(T) (16S rRNA gene sequence similarity, 99.0 %) and Pseudoxanthomonas sacheonensis KCTC 22080(T) (98.0 %). The levels of 16S rRNA gene sequence similarity with respect to other Pseudoxanthomonas species with validly published names were less than 96.5 %. DNA-DNA relatedness values for strain ZFJR-3(T) with respect to its closely related neighbours P. yeongjuensis KCTC 22757(T) and P. sacheonensis KCTC 22080(T) were 48.7 and 36.3 %, respectively. Based on the phenotypic, phylogenetic and genotypic data, strain ZFJR-3(T) is considered to represent a novel species of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas gei sp. nov. is proposed. The type strain is ZFJR-3(T) (=CCTCC AB 2013020(T) =KCTC 32298(T)).

Solirubrobacter phytolaccae sp. nov., an endophytic bacterium isolated from roots of Phytolacca acinosa Roxb.

A Gram-staining-positive, strictly aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated GTGR-8(T), which formed white colonies, was isolated from roots of Phytolacca acinosa Roxb. collected from Taibai Mountain in Shaanxi Province, north-west China. Strain GTGR-8(T) grew optimally at 28-30 °C, at pH 7.0-8.0 and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GTGR-8(T) was a member of the genus Solirubrobacter and was closely related to Solirubrobacter pauli B33D1(T) (98.9% similarity), Solirubrobacter ginsenosidimutans BXN5-15(T) (97.0%) and Solirubrobacter soli Gsoil 355(T) (96.9%). No other recognized bacterial species showed more than 94.2% 16S rRNA gene sequence similarity to the novel isolate. The only respiratory quinone of strain GTGR-8(T) was MK-7(H4) and the major fatty acids (>5%) were iso-C16 : 0, C18 : 1ω9c, C17 : 1ω8c, C18 : 3ω6c (6,9,12) and C17 : 1ω6c. The DNA G+C content was 71.0 mol%. DNA-DNA relatedness for strain GTGR-8(T) with respect to its closest relatives, S. pauli KCTC 9974(T) and S. ginsenosidimutans KCTC 19420(T), was 52.5 and 24.5%, respectively. Based on phenotypic, phylogenetic and genotypic data, strain GTGR-8(T) is considered to represent a novel species in the genus Solirubrobacter, for which the name Solirubrobacter phytolaccae sp. nov. is proposed. The type strain is GTGR-8(T) ( = CCTCC AB 2013011(T) = KCTC 29190(T)).

Pontibacter toksunensis sp. nov., isolated from soil, and emended descriptions of Pontibacter roseus and Pontibacter akesuensis.

A Gram-reaction-negative, rod-shaped, non-motile, red-pigmented bacterial strain, designated ZLD-7(T), was isolated from a soil sample collected from an arid area in Xinjiang Province in north-west China, and characterized by using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis showed that strain ZLD-7(T) was a member of the genus Pontibacter in the family Cytophagaceae, with sequence similarities of 93.7-96.2 % to type strains of other Pontibacter species. The only isoprenoid quinone of strain ZLD-7(T) was MK-7, and its major cellular fatty acids were summed feature 4 (anteiso-C17 : 1 B and/or iso-C17 : 1 I), iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The polar lipid profile consisted of phosphatidylethanolamine, one unidentified aminolipid and four unidentified polar lipids. The DNA G+C content was 47.8 mol%. On the basis of the evidence presented, it is proposed that strain ZLD-7(T) represents a novel species of the genus Pontibacter, for which the name Pontibacter toksunensis sp. nov. is proposed. The type strain is ZLD-7(T) ( = CCTCC AB 208003(T) = KCTC 23984(T)). Emended descriptions of Pontibacter roseus and Pontibacter akesuensis are also proposed.

Taibaiella smilacinae gen. nov., sp. nov., an endophytic member of the family Chitinophagaceae isolated from the stem of Smilacina japonica, and emended description of Flavihumibacter petaseus.

A light-yellow-coloured bacterium, designated strain PTJT-5(T), was isolated from the stem of Smilacina japonica A. Gray collected from Taibai Mountain in Shaanxi Province, north-west China, and was subjected to a taxonomic study by using a polyphasic approach. The novel isolate grew optimally at 25-28 °C and pH 6.0-7.0. Flexirubin-type pigments were produced. Cells were Gram-reaction-negative, strictly aerobic, rod-shaped and non-motile. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PTJT-5(T) was a member of the phylum Bacteroidetes, exhibiting the highest sequence similarity to Lacibacter cauensis NJ-8(T) (87.7 %). The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 and iso-C17 : 0 3-OH. The only polyamine was homospermidine and the major polar lipid was phosphatidylethanolamine. The only respiratory quinone was MK-7 and the DNA G+C content was 40.3 mol%. Based on the phenotypic, phylogenetic and genotypic data, strain PTJT-5(T) is considered to represent a novel species of a new genus in the family Chitinophagaceae, for which the name Taibaiella smilacinae gen. nov., sp. nov. is proposed. The type strain of Taibaiella smilacinae is PTJT-5(T) ( = CCTCC AB 2013017(T) = KCTC 32316(T)). An emended description of Flavihumibacter petaseus is also proposed.

[Correlation between expression of HIF-2α and OCT-4 and prognosis of NSCLC].

OBJECTIVE: To investigate the expression and significance of hypoxia inducible factor-2α (HIF-2α) and transcription factor OCT-4 in non-small cell lung cancer (NSCLC), and evaluate their roles in the prognosis of NSCLC. METHODS: Tissues from 51 cases of NSCLC were collected and immunohistochemistry (SP method) was used to detect the expression of HIF-2α and OCT-4 proteins. The correlation between the protein expression and the prognosis of NSCLC was analyzed. RESULTS: The positive rates of HIF-2α and OCT-4 expression in the NSCLC were 52.9% and 72.5%, respectively. There was significant relation between the expression of HIF-2α and OCT-4 (r=0.514,P<0.01). High expression of them revealed poor prognosis for NSCLC patients characterized with a bad overall survival(P<0.05). CONCLUSION: There is a negative corelation between the expression of HIF-2α and OCT-4 and the prognosis of NSCLC. Combined examination of HIF-2α and OCT-4 expression might be an important biomarker for NSCLC prognosis.