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 silico analysis of Simple Sequence Repeats from chloroplast genomes of Solanaceae species

The availability of chloroplast genome (cpDNA) sequences of Atropa belladonna, Nicotiana sylvestris, N.tabacum, N. tomentosiformis, Solanum bulbocastanum, S. lycopersicum and S. tuberosum, which are Solanaceae species,allowed us to analyze the organization of cpSSRs in their genic and intergenic regions. In general, the number of cpSSRs incpDNA ranged from 161 in S. tuberosum to 226 in N. tabacum, and the number of intergenic cpSSRs was higher than geniccpSSRs. The mononucleotide repeats were the most frequent in studied species, but we also identified di-, tri-, tetra-, pentaandhexanucleotide repeats. Multiple alignments of all cpSSRs sequences from Solanaceae species made the identification ofnucleotide variability possible and the phylogeny was estimated by maximum parsimony. Our study showed that the plastomedatabase can be exploited for phylogenetic analysis and biotechnological approaches

Disruption of the ORFs XF-810, XF-818, XF-1940, XF-2359 and XF-2708 of Xylella fastidiosa-CVC probably related to pathogenicity of the bacteria

Clorose variegada dos citros (CVC), também conhecida como amarelinho, é uma das doenças de citros mais severas da citricultura brasileira. A CVC é causada pela bactéria limitada ao xilema Xylella fastidiosa. Esta bactéria afeta principalmente as variedades de laranja doce e foi relatada pela primeira vez em 1987 nos Estados de São Paulo e Minas Gerais. O genoma de X. fastidiosa linhagem 9a5c foi completamente seqüenciado e revelou muitos genes provavelmente envolvidos na patogenicidade desta bactéria. Entre estes genes estão as endoglicanases, que são enzimas degradadoras de componentes da parede celular vegetal do hospedeiro e as adesinas, que são proteínas envolvidas na adesão das células bacterianas no xilema, como também auxiliam na formação de agregados bacterianos que causam a obstrução dos vasos do xilema, interferindo no transporte de água e sais minerais para todas as partes da planta, levando ao aparecimento dos sintomas relacionados a CVC. O objetivo deste trabalho foi a obtenção de mutantes de X. fastidiosa linhagem J1a12, pela disrupção das ORFs Xf-810, Xf-818 e Xf-2708, que apresentam função putativa de endoglicanases, da ORF Xf-2359 possivelmente relacionada à uma pectate liase e da ORF Xf-1940, que apresenta similaridade de seqüência à metionina sulfoxido redutase, proteína identificada com a função de possuir adesão funcional na superfície da célula bacteriana. Neste trabalho, duas estratégias foram utilizadas para a construção dos vetores de disrupção das ORFs selecionadas no genoma de X. fastidiosa: (1) substituição das ORFs pelo gene yfp, que codifica a proteína fluorescente amarela e, (2) inserção do cassete lacZ-KamR interrompendo a região codificadora das ORFs. Estes vetores foram usados para transformar X. fastidiosa, linhagem J1a12 por eletroporação e confirmou-se a obtenção das bactérias mutantes por reações em cadeia da polimerase (PCR) com oligonucleotídeos específicos.Variegated Clorose of the citros (CVC), also known as "amarelinho", is one of the more severe diseases of citrus of the Brazilian Citrus plantation. CVC is caused by Xylella fastidiosa, a limited bacteria to the xylem. This bacterium affects mainly sweet orange varieties and it was reported for the first time in 1987 in the states of São Paulo and Minas Gerais. The genoma of X. fastidiosa strain 9a5c was completely sequenced and it revealed many genes probably involved in the patogenicity of the bacteria. Among the genes, are the endoglucanases, that are degradative enzymes of the host cell wall components and the adesins, that are proteins involved in the adhesion of the bacterial cells in the xylem, as well as they aid in the formation of bacterial aggregation that cause the obstruction of the xylem vessels interfering in the transport of water and mineral salts for all the parts of the plant, providing the emergence of the symptoms related to CVC. The objective of this work was to produce mutants of X. fastidiosa strain J1a12, by disruption of the ORFs Xf-810, Xf-818 and Xf-2708, that present putative function of endoglucanases, Xf-2359 possibly related to a pectate liase and of Xf-1940, that presents sequence similarity to the methionine sulfoxide reductase, identified protein with the functional function of adhesion in the surface of the bacterial cell. In this work, two strategies were used for the construction of the disruption vectors of the selected ORFs in the X. fastidiosa genome: (1) substitution of the ORFs for the yfp gene, that codifies for the yellow fluorescent protein and, (2) insertion of the lacZ-KamR cassette into the coding region of the ORFs. These vectors were used to transform X. fastidiosa strain J1a12 by electroporation and the mutants produced were confirmed by polymerase chain reactions (PCR) with specific oligonucleotids

Disruption of the ORFs XF-810, XF-818, XF-1940, XF-2359 and XF-2708 of Xylella fastidiosa-CVC probably related to pathogenicity of the bacteria

Clorose variegada dos citros (CVC), também conhecida como amarelinho, é uma das doenças de citros mais severas da citricultura brasileira. A CVC é causada pela bactéria limitada ao xilema Xylella fastidiosa. Esta bactéria afeta principalmente as variedades de laranja doce e foi relatada pela primeira vez em 1987 nos Estados de São Paulo e Minas Gerais. O genoma de X. fastidiosa linhagem 9a5c foi completamente seqüenciado e revelou muitos genes provavelmente envolvidos na patogenicidade desta bactéria. Entre estes genes estão as endoglicanases, que são enzimas degradadoras de componentes da parede celular vegetal do hospedeiro e as adesinas, que são proteínas envolvidas na adesão das células bacterianas no xilema, como também auxiliam na formação de agregados bacterianos que causam a obstrução dos vasos do xilema, interferindo no transporte de água e sais minerais para todas as partes da planta, levando ao aparecimento dos sintomas relacionados a CVC. O objetivo deste trabalho foi a obtenção de mutantes de X. fastidiosa linhagem J1a12, pela disrupção das ORFs Xf-810, Xf-818 e Xf-2708, que apresentam função putativa de endoglicanases, da ORF Xf-2359 possivelmente relacionada à uma pectate liase e da ORF Xf-1940, que apresenta similaridade de seqüência à metionina sulfoxido redutase, proteína identificada com a função de possuir adesão funcional na superfície da célula bacteriana. Neste trabalho, duas estratégias foram utilizadas para a construção dos vetores de disrupção das ORFs selecionadas no genoma de X. fastidiosa: (1) substituição das ORFs pelo gene yfp, que codifica a proteína fluorescente amarela e, (2) inserção do cassete lacZ-KamR interrompendo a região codificadora das ORFs. Estes vetores foram usados para transformar X. fastidiosa, linhagem J1a12 por eletroporação e confirmou-se a obtenção das bactérias mutantes por reações em cadeia da polimerase (PCR) com oligonucleotídeos específicos.Variegated Clorose of the citros (CVC), also known as "amarelinho", is one of the more severe diseases of citrus of the Brazilian Citrus plantation. CVC is caused by Xylella fastidiosa, a limited bacteria to the xylem. This bacterium affects mainly sweet orange varieties and it was reported for the first time in 1987 in the states of São Paulo and Minas Gerais. The genoma of X. fastidiosa strain 9a5c was completely sequenced and it revealed many genes probably involved in the patogenicity of the bacteria. Among the genes, are the endoglucanases, that are degradative enzymes of the host cell wall components and the adesins, that are proteins involved in the adhesion of the bacterial cells in the xylem, as well as they aid in the formation of bacterial aggregation that cause the obstruction of the xylem vessels interfering in the transport of water and mineral salts for all the parts of the plant, providing the emergence of the symptoms related to CVC. The objective of this work was to produce mutants of X. fastidiosa strain J1a12, by disruption of the ORFs Xf-810, Xf-818 and Xf-2708, that present putative function of endoglucanases, Xf-2359 possibly related to a pectate liase and of Xf-1940, that presents sequence similarity to the methionine sulfoxide reductase, identified protein with the functional function of adhesion in the surface of the bacterial cell. In this work, two strategies were used for the construction of the disruption vectors of the selected ORFs in the X. fastidiosa genome: (1) substitution of the ORFs for the yfp gene, that codifies for the yellow fluorescent protein and, (2) insertion of the lacZ-KamR cassette into the coding region of the ORFs. These vectors were used to transform X. fastidiosa strain J1a12 by electroporation and the mutants produced were confirmed by polymerase chain reactions (PCR) with specific oligonucleotids

Identification of genes involved in defense against pathogens in the CitEST databank and in macroarrays of Citrus sinensis-Guignardia citricarpa interaction

A citricultura brasileira concentra-se principalmente no Estado de São Paulo que contribui com 80,4 % da produção nacional, sendo o Brasil um dos maiores produtores mundiais de citros. Um dos problemas enfrentados pela citricultura é a sua vulnerabilidade a pragas e doenças, devido principalmente a baixa diversidade genética nas variedades comerciais utilizadas, associada ao sistema de plantio em áreas extensas. Uma das doenças que vem causando crescentes prejuízos para a citricultura brasileira é a pinta preta ou mancha preta dos citros causada pelo fungo Guignardia citricarpa Kiely. O uso de conhecimentos de biologia molecular e métodos biotecnológicos devem ser considerados como importante alternativa para a produção de plantas geneticamente modificadas expressando genes de resistência. Para se obter plantas de citros resistentes a doenças, se faz necessário identificar genes que estejam relacionados com os mecanismos de defesa da planta. Na tentativa de identificar estes genes, o objetivo geral deste trabalho foi a identificação de genes in silico no banco de dados do Projeto Millenium CitEST e a análise de expressão diferencial de genes envolvidos na defesa. Mais de 7600 sequencias foram identificadas nas buscas no CitEST com similaridade aos genes R e genes envolvidos na HR e defesa, MAPKs e SNF1. Destes, foram selecionados 273 sequencias para experimentos de macroarranjo para análise da interação Citrus sinensis-Guignardia citricarpa. A análise estatística revelou que 171 genes (62,63%) apresentaram expressão diferencial significativa ao nível de 5% de probabilidade. Destes, 80 apresentaram expressão diferencial significativa maior do que duas vezes, dos quais 38 genes foram induzidos e 42 foram reprimidos no tecido infectado. Entre os genes induzidos estão MAPKs, genes de resistência (R), genes envolvidos na resposta de hipersensibilidade (HR) e na defesa da planta. Entre os transcritos reprimidos, há quatro similares a peroxidases e cinco similares a catalases, o que era esperado já que catalases e algumas peroxidases são capazes de remover H2O2, e assim a planta produz espécies reativa de oxigênio capaz de desencadear a ativação de genes de defesa. Os dados do macroarranjo foram validados via transcrição reversa seguida de PCR quantitativo em tempo real (RT-PCRq) de 9 genes. As análises confirmaram a expressão diferencial de 8 deles sendo que somente um apresentou resultado contrastante ao macroarranjo, o que demonstra a eficiência da metodologia de macroarranjos para estudo de muitos genes simultaneamente. Os genes diferencialmente expressos identificados na interação C. sinensis-G. citricarpa são de grande importância, pois são fortes candidatos para serem utilizados na transformação genética de plantas com o objetivo de obter novas variedades de plantas com resistência a patógenos.The Brazilian citrus industry is concentrated mainly in the State of Sao Paulo which contributes with 80.4% of national production, with Brazil being a leading world producer of citrus. One of the problems facing the citrus industry is its vulnerability to pests and diseases, mainly due to low genetic diversity of the commercial varieties used, linked to the system of planting in extensive areas. A disease that is causing increasing damage to the brazilian citrus industry is the black spot of citrus caused by the fungus Guignardia citricarpa Kiely. The use of knowledge of molecular biology and biotechnological methods should be considered as an important alternative for the production of genetically modified plants expressing genes for resistance. In order to obtain citrus plants resistant to diseases it is necessary to identify genes that are related to the defense mechanisms of the plant. In an attempt to identify these genes, the general aim of this study was to identify genes in silico in the database of the Millennium CitEST Project and to perform differential expression analysis of genes involved in the defense mechanisms. More than 7600 reads were identified in the CitEST search with similarity to R genes, genes involved in HR and defense, MAPKs and SNF1. It was selected 273 reads for macroarray experiments to analysis of Citrus sinensis-Guignardia citricarpa interaction. Statistical analysis revealed that 171 genes (62.63%) showed significant differential expression at the level of 5% probability. From these, 80 showed significant differential expression higher than two fold, in which 38 genes were induced and 42 were repressed in infected tissue. Among the induced genes are MAPKs, resistance (R) genes, genes involved in hypersensitivity response (HR) and plant defense. Among the suppressed transcripts, there are four similar to peroxidases and five similar to catalases, which is expected because catalases and some peroxidases are able to remove H2O2, and so the plant produces reactive oxygen species capable of triggering the activation of defense genes. The macroarray data were validated by reverse transcription followed by quantitative real-time PCR (RT-PCRq) of 9 genes. The analysis confirmed the differential expression of 8 of them, and only one presented different result of macroarray which demonstrate the efficiency of the macroarray methodology to analyze several genes simultaneously. The genes differentially expressed in the interaction of C. sinensis x Guignardia citricarpa identified are of great importance because they are strong candidates for use in genetic transformation of plants with the objective of obtaining new varieties of plants resistant to pathogens

Identification of protein kinase SNF1 in CitEST

SnRKs (Sucrose non-fermenting-1 related kinases) is a family of protein kinases found in many crops, such as Arabidopsis, rice, sugarcane, tomato and several other plant species. This family of proteins is also present in other organisms like Saccharomyces cerevisiae (sucrose non-fermenting-1 - Snf1) and in mammals (AMP-activated protein kinases - AMPKs). There is evidence that SnRKs play an important role in plant responses to nutritional and environmental stresses and that SnRKs also play a major role in controlling key enzymes in the biosynthetic pathways of plants. In this work, we identified 18 contigs and two singletons encoding putative SnRKs in the CitEST database. All of them present highly conserved N-terminal catalytic domain, which is found in the SnRKs families of several plant species. Through comparison with known SnRKs, we were able to classify them into three subfamilies