Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus andCortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatantsof termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensionalgel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.Fil: Ben Guerrero, Emiliano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Arneodo Larochette, Joel Demián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Bombarda Campanha, Raquel. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Oliveira, Patrícia Abrão de. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Labate, Mônica T. Veneziano. Universidade de Sao Paulo; BrasilFil: Cataldi, Thaís Regiani. Universidade de Sao Paulo; BrasilFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Carlos A.. Universidade de Sao Paulo; BrasilFil: Rodrigues, Clenilson Martins. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Talia, Paola Monica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Cell Wall Proteomics as a Means to Identify Target Genes to Improve Second‐Generation Biofuel Production

Second‐generation biofuels (B2G) generally uses residues composed of lignocellulosic materials to produce renewable energy (potentially up to 50%), without increasing the planted areas. However, the high cost of enzymes required for cell wall disassembly prior to the saccharification makes the B2G production more expensive yet, compared to the first‐generation biofuels. Designing plants with less lignin, a barrier to B2G production, or facilitating cell wall disassembly by searching for the plant mechanisms can be the way to obtain B2G feasibility. Therewith, plant cell wall proteomics provides valuable information concerning the main cell wall proteins (CWPs) involved in its biosynthesis and rearrangements. Essentially, two plants of the grass family have been studied: sugarcane as a crop amenable to second‐generation ethanol (E2G) production; and Brachypodium distachyon as a model plant amenable to genetic transformation. Cell wall proteomics has allowed the identification of numerous CWPs as well as their fine profiling in different organs and at various developmental stages. Proteins acting on carbohydrates, mostly glycosyl hydrolases, and oxidoreductases, including class III peroxidases and laccases, can be highlighted. Both kinds of CWPs are assumed to contribute to the remodelling of cell wall polysaccharides by enzymatic or non‐enzymatic mechanisms. CWPs present in growing organs could also be attractive candidates since they greatly contribute to cell wall plasticity

Analysis of the biofilm proteome of Xylella fastidiosa

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa </it>is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of <it>X. fastidiosa </it>strain 9a5c, in comparison to planktonic growth condition.</p> <p>Results</p> <p>We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of <it>X. fastidiosa</it>. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments.</p> <p>Conclusions</p> <p>We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of <it>X. fastidiosa</it>, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.</p

Modulating MIOX2 expression in Nicotiana tabacum and impacts on genes involved in cell wall biosynthesis

Cell walls are essential structures for plant development and growth. Apart from its biological functions, the polysaccharides that make cell walls (cellulose, hemicellulose and pectins) are the principal natural fibrous materials, considered the most important renewable resource on earth, used as raw material for many industrial processes among them, for pulp and paper production, charcoal, and biofuels. For all these reasons, the study of molecular composition and biosynthesis of plant cell walls has been a matter of great interest for researchers over the past few years. In this context, a full-length cDNA fragment of Miox2 gene was cloned from Arabidopsis seedlings, using RT-PCR, with an open reading frame of 954 pb and a corresponding protein subunit molecular mass of 37 kDa. The deduced amino acid sequence of the cDNA showed a high degree of homology with myo-Inosytol oxygenases from other organisms. This cDNA was used for genetic transformation of model plants (tobacco), which expressed either antisense or sense RNA. Transgenic homozygous tobacco model plants with either repression or constitutively expressed Miox2 were obtained with the number of copies varying from 1 to 7. Neither, the repression of the endogenous tobacco Miox genes or the constitutive expression of Miox2 gene, caused major impact on plant development, leaf morphology or flowering time. There was however, statistically significant (P<0.05) changes in the arabinan and D-galacturonate contents. These results clearly indicate that the modulation of the myo-Inositol pathway caused no major impact on cell wall polysaccharide biosynthesis.As paredes celulares vegetais são estruturas essenciais para o crescimento e desenvolvimento das plantas. Além de suas diversas funções biológicas, os componentes polissacarídicos constituintes das paredes celulares (celulose, hemiceluloses e pectinas) são de vital importância como fonte natural de fibras, sendo consideradas as fontes principais de recursos renováveis do planeta, utilizados como matéria prima para diversos processos industriais, dentre eles, a produção de papel e celulose, carvão vegetal e biocombustíveis. Todos esses fatores têm despertado grande interesse no estudo da composição e biossíntese das paredes celulares. Neste contexto, um fragmento de cDNA do gene Miox2 foi clonado de plântulas de Arabidopsis, via RT-PCR, com uma região aberta de leitura de 954 pb e sua proteína com massa molecular de 37kDa. A sequência deduzida de aminoácidos do cDNA apresentou alto grau de identidade com mio-Inositol oxigenases de outros organismos. Este cDNA foi usado para transformação genética de plantas modelo (tabaco) que produziram RNA antisense ou sense. Plantas de tabaco homozigotas para o transgene com repressão ou expressão constitutiva do gene Miox2 foram obtidas com um número de cópias do transgene, variando de 1 a 7. A repressão do gene Miox de tabaco endógeno assim como a expressão constitutiva do gene Miox2 de Arabidopsis não causaram alterações no desenvolvimento, morfologia foliar ou tempo de florescimento das plantas. Entretanto, alterações estatisticamente significativas (P<0.05) ocorreram no conteúdo de arabinana e de D-galacturonato. Estes resultados indicam que a modulação do metabolismo do mio-Inositol não causou grandes impactos na biossíntese dos polissacarídeos da parede celular.Fil: Defávari Nascimento, D.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Conti, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Mônica T. V.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Gutmanis, Gunta. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bertolo, Ana L. F.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: de Andrade, Alexander. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Bragatto, Juliano. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Pagotto, Luís Otávio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Damin, Plínio. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Moon, David H.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; BrasilFil: Labate, Carlos A.. Universidade do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz; Brasi

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms\ud associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular\ud mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression\ud have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer\ud after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell\ud carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild\ud type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass\ud spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed\ud using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array\ud showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX\ud overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein\ud expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM\ud and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of\ud microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate\ud actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced\ud cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated\ud genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.FAPESPCNP

ProbMetab: an R package for Bayesian probabilistic annotation of LC-MS based metabolomics

We present ProbMetab, an R package which promotes substantial improvement in automatic probabilistic LC-MS based metabolome annotation. The inference engine core is based on a Bayesian model implemented to: (i) allow diverse source of experimental data and metadata to be systematically incorporated into the model with alternative ways to calculate the likelihood function and; (ii) allow sensitive selection of biologically meaningful biochemical reactions databases as Dirichlet-categorical prior distribution. Additionally, to ensure result interpretation by system biologists, we display the annotation in a network where observed mass peaks are connected if their candidate metabolites are substrate/product of known biochemical reactions. This graph can be overlaid with other graph-based analysis, such as partial correlation networks, in a visualization scheme exported to Cytoscape, with web and stand alone versions. ProbMetab was implemented in a modular fashion to fit together with established upstream (xcms, CAMERA, AStream, mzMatch.R, etc) and downstream R package tools (GeneNet, RCytoscape, DiffCorr, etc). ProbMetab, along with extensive documentation and case studies, is freely available under GNU license at: http://labpib.fmrp.usp.br/methods/probmetab/.Comment: Manuscript to be submitted very soon. 7 pages, 3 color figures. There is a companion material, the two case studies, which are going to be posted here together with the main text in next updated versio

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo - FAPESP [04/11799-0, 2008/52691-9]Fundacsao de Apoio a Pesquisa do Estado de Sao Paulo FAPES

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract\ud \ud \ud \ud Background\ud \ud In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM).\ud \ud \ud \ud Results\ud \ud Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones.\ud \ud \ud \ud Conclusion\ud \ud Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.The authors are grateful to FAPESP and CNPq for the financial support for this work via grants # 2010/11135-6; 2009/18354-8; 2010/08370-3; 2008/ 56255-9 and 2010/52362-5 (FAPESP); grants # 159341/2011-6; 482166/ 2010-0 and 490022/2009-0 (CNPq), Projeto INCT do Bioetanol (CNPq/ FAPESP), to USP for the financial via NAP Centro de Instrumentação para Estudos Avançados de Materiais Nanoestruturados e Biossistemas and NAP de Bioenergia e Sustentabilidade, and European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement n° 251132. We are also grateful to Valeria Gazda for the help with furfural and 5-HMF chromatographic analysis and Caragh Whitehead for the paper review. The electron microscopy work has been performed on the microscopes JSM-5900LV and Quanta 650-FEG at LME/LNNano/ CNPEM, Campinas

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production - part 1

Background: In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass fromgrass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results: Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion: Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.FAPESP (10/11135-6, 09/18354-8, 10/08370-3, 08/56255-9, 10/52362-5)CNPq (159341/2011-6, 482166/2010-0, 490022/2009-0)European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013)INCT do BioetanolNúcleo de Apoio à Pesquisa em Bioenergia e Sustentabilidade (NAPBS)Centro de Instrumentação para Estudos Avançados de Materiais Nanoestruturados e Biossistemas (CIEA_MNB