Detecting Network Communities: An Application to Phylogenetic Analysis

This paper proposes a new method to identify communities in generally weighted complex networks and apply it to phylogenetic analysis. In this case, weights correspond to the similarity indexes among protein sequences, which can be used for network construction so that the network structure can be analyzed to recover phylogenetically useful information from its properties. The analyses discussed here are mainly based on the modular character of protein similarity networks, explored through the Newman-Girvan algorithm, with the help of the neighborhood matrix . The most relevant networks are found when the network topology changes abruptly revealing distinct modules related to the sets of organisms to which the proteins belong. Sound biological information can be retrieved by the computational routines used in the network approach, without using biological assumptions other than those incorporated by BLAST. Usually, all the main bacterial phyla and, in some cases, also some bacterial classes corresponded totally (100%) or to a great extent (>70%) to the modules. We checked for internal consistency in the obtained results, and we scored close to 84% of matches for community pertinence when comparisons between the results were performed. To illustrate how to use the network-based method, we employed data for enzymes involved in the chitin metabolic pathway that are present in more than 100 organisms from an original data set containing 1,695 organisms, downloaded from GenBank on May 19, 2007. A preliminary comparison between the outcomes of the network-based method and the results of methods based on Bayesian, distance, likelihood, and parsimony criteria suggests that the former is as reliable as these commonly used methods. We conclude that the network-based method can be used as a powerful tool for retrieving modularity information from weighted networks, which is useful for phylogenetic analysis

Construction and validation of metagenomic DNA libraries from landfarm soil microorganisms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Landfarming biodegradation is a strategy used by the petrochemical industry to reduce pollutants in petroleum-contaminated soil. We constructed 2 metagenomic libraries from landfarming soil in order to determine the pathway used for mineralization of benzene and to examine protein expression of the bacteria in these soils. The DNA of landfarm soil, collected from Ilheus, BA, Brazil, was extracted and a metagenomic library was constructed with the Copy Control (TM) Fosmid Library Production Kit, which clones 25-45-kb DNA fragments. The clones were selected for their ability to express enzymes capable of cleaving aromatic compounds. These clones were grown in Luria-Bertani broth plus L-arabinose, benzene, and chloramphenicol as induction substances; they were tested for activity in the catechol cleavage pathway, an intermediate step in benzene degradation. Nine clones were positive for ortho-cleavage and one was positive for meta-cleavage. Protein band patterns determined by SDS-polyacrylamide gel electrophoresis differed in bacteria grown on induced versus non-induced media (Luria-Bertani broth). We concluded that the DNA of landfarm soil is an important source of genes involved in mineralization of xenobiotic compounds, which are common in gasoline and oil spills. Metagenomic library allows identification of non-culturable microorganisms that have potential in the bioremediation of contaminated sites.12221482155Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNPq [558272/2009-6

Carbon source-induced changes in the physiology of the cacao pathogen Moniliophthora perniciosa (Basidiomycetes) affect mycelial morphology and secretion of necrosis-inducing proteins

Quantitative and qualitative relationships were found between secreted proteins and their activity, and the hyphal morphology of Moniliophthora perniciosa, the causal agent of witches' broom disease in Theobroma cacao. This fungus was grown on fermentable and non-fermentable carbon sources; significant differences in mycelial morphology were observed and correlated with the carbon source. A biological assay performed with Nicotiana tabacum leaves revealed that the necrosis-related activity of extracellular fungal proteins also differed with carbon source. There were clear differences in the type and quantity of the secreted proteins. In addition, the expression of the cacao molecular chaperone BiP increased after treatment with secreted proteins, suggesting a physiological response to the fungus secretome. We suggest that the carbon source-dependent energy metabolism of M. perniciosa results in physiological alterations in protein expression and secretion; these may affect not only M. perniciosa growth, but also its ability to express pathogenicity proteins.8310351050FINEP/FAPES

Hydrogen peroxide formation in cacao tissues infected by the hemibiotrophic fungus Moniliophthora perniciosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)In plant pathogen interaction, the hydrogen peroxide (H(2)O(2)) may play a dual role: its accumulation inhibits the growth of biotrophic pathogens, while it could help the infection/colonization process of plant by necrotrophic pathogens. One of the possible pathways of H(2)O(2) production involves oxalic acid (Oxa) degradation by apoplastic oxalate oxidase. Here, we analyzed the production of H(2)O(2), the presence of calcium oxalate (CaOx) crystals and the content of Oxa and ascorbic acid (Asa) the main precursor of Oxa in plants in susceptible and resistant cacao (Theobroma cacao L) infected by the hemibiotrophic fungus Moniliophthora perniciosa. We also quantified the transcript level of ascorbate peroxidase (Apx), germin-like oxalate oxidase (Gip) and dehydroascorbate reductase (Dhar) by RT-qPCR. We report that the CaOx crystal amount and the H(2)O(2) levels in the two varieties present distinct temporal and genotype-dependent patterns. Susceptible variety accumulated more CaOx crystals than the resistant one, and the dissolution of these crystals occurred in the early infection steps and in the final stage of the disease in the resistant and the susceptible variety, respectively. High expression of the Glp and accumulation of Oxa were observed in the resistant variety. The content of Asa increased in the inoculated susceptible variety, but remained constant in the resistant one. The susceptible variety presented reduced Dhar expression. The role of H(2)O(2) and its formation from Oxa via Apx and Glp in resistant and susceptible variety infected by M. perniciosa were discussed. (C) 2011 Elsevier Masson SAS. All rights reserved.498917922Financiadora de Estudos e Projetos (FINEP) [01.07.0074-00]Fundacao de Amparo a Pesquisa da Bahia (FAPESB) [1431080017116]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP) [01.07.0074-00]Fundacao de Amparo a Pesquisa da Bahia (FAPESB) [1431080017116

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. in order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction - under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF - using infiltration-centrifugation procedure - that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.29112391240

Identification and Characterization of a Class III Chitin Synthase Gene of Moniliophthora perniciosa, the Fungus That Causes Witches' Broom Disease of Cacao

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Chitin synthase (CHS) is a glucosyltransferase that converts UDP-N-acetylglucosamine into chitin, one of the main components of fungal cell wall. Class III chitin synthases act directly in the formation of the cell wall. They catalyze the conversion of the immediate precursor of chitin and are responsible for the majority of chitin synthesis in fungi. As such, they are highly specific molecular targets for drugs that can inhibit the growth and development of fungal pathogens. In this work, we have identified and characterized a chitin synthase gene of Moniliophthora perniciosa (Mopchs) by primer walking. The complete gene sequence is 3,443 bp, interrupted by 13 small introns, and comprises a cDNA with an ORF with 2,739 bp, whose terminal region was experimentally determined, encoding a protein with 913 aa that harbors all the motifs and domains typically found in class III chitin synthases. This is the first report on the characterization of a chitin synthase gene, its mature transcription product, and its putative protein in basidioma and secondary mycelium stages of M. perniciosa, a basidiomycotan fungus that causes witches' broom disease of cacao.474431440Fundacao de Amparo a Pesquisa do Estado da Bahia (FAPESB)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Expression of an Oxalate Decarboxylase Impairs the Necrotic Effect Induced by Nep1-like Protein (NLP) of Moniliophthora perniciosa in Transgenic Tobacco

Oxalic acid (OA) and Nep1-like proteins (NLP) are recognized as elicitors of programmed cell death (PCD) in plants, which is crucial for the pathogenic success of necrotrophic plant pathogens and involves reactive oxygen species (ROS). To determine the importance of oxalate as a source of ROS for OA- and NLP-induced cell death, a full-length cDNA coding for an oxalate decarboxylase (FvOXDC) from the basidiomycete Flammulina velutipes, which converts OA into CO(2) and formate, was overexpressed in tobacco plants. The transgenic plants contained less OA and more formic acid compared with the control plants and showed enhanced resistance to cell death induced by exogenous OA and MpNEP2, an NLP of the hemibiotrophic fungus Moniliophthora perniciosa. This resistance was correlated with the inhibition of ROS formation in the transgenic plants inoculated with OA, MpNEP2, or a combination of both PCD elicitors. Taken together, these results have established a pivotal function for oxalate as a source of ROS required for the PCD-inducing activity of OA and NLP. The results also indicate that FvOXDC represents a potentially novel source of resistance against OA- and NLP-producing pathogens such as M. perniciosa, the causal agent of witches' broom disease of cacao (Theobroma cacao L.).247839848Fundacao de Amparo a Pesquisa do Estado da Bahia (Salvador, Brazil)National Council for Scientific and Technological Development (Brasilia, Brazil)Coordination of Higher Education and Graduate Training (Brasilia, Brazil