AVALIAÇÃO DO IMPACTO DO LODO DE ESGOTO NA MICROBIOTA DO SOLO UTILIZANDO O GENE 16S rRNA

ABSTRACT The objective of this work was to evaluate and compare the microbial diversity of a soil treated with sewage sludge (BAR 1N) with the same soil without treatment (control). The use of industrial and domestic sewer sludge as organic fertilizer in agricultural soils is currently considered a promising alternative for the final destination of these residues. Molecular studies using the analysis of the gene 16S rRNA provide relevant information in regard to studies of microbial ecology, since it is believed that only 10% of these microorganisms can be cultivated. The sequences were submitted to analysis of nucleotide similarity, based on data in GenBank, in order to be identified and classified. Following phylogram analysis, a high number of nonidentified microorganisms were observed in the investigated soils. The results demonstrated that the outstanding bacterial phyla were Acidobacteria and Proteobacteria. Phylogenetic analyses revealed differences in both soils, based on the bacterial diversity index, showing that the test soil had more diversity when compared to the BAR 1N soil.

RESUMO Este trabalho teve por objetivo estimar e comparar a diversidade microbiana de um solo tratado com lodo de esgoto (BAR 1N) com o mesmo solo sem tratamento (controle). A utilização do lodo de esgoto de origem industrial ou domiciliar em solos agrícolas como adubo orgânico é considerado, atualmente, uma alternativa promissora para disposição final deste resíduo. Estudos moleculares que utilizam a análise do gene 16S rRNA permitem a obtenção de informações relevantes acerca da ecologia microbiana, pois acredita-se que apenas 10% desses microorganismos podem ser cultivados. O DNA genômico dos micro-organismos presentes em ambos os solos foi extraído, clonado e, após amplificação por PCR, foi feito o sequenciamento do gene 16S rRNA. As sequências obtidas foram submetidas à análise de similaridade de nucleotídeos com o banco de dados GenBank para que pudessem ser identificadas e classificadas. Após a análise dos filogramas observou-se um número elevado de micro-organismos não identificados nos solos analisados. Os resultados demonstraram que os filos bacterianos que se destacaram foram Acidobacteria e Proteobacteria. Análises filogenéticas revelaram diferenças entre os solos, mostrando por meio de índice de diversidade bacteriana que o solo controle apresentou maior diversidade quando comparado ao solo BAR 1N. O filo Nitrospira revelou-se significativamente afetado pela aplicação do lodo de esgoto.

Optimal clone identifier for genomic shotgun libraries: "OC Identifier tool".

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray.

Optimal clone identifier for genomic shotgun libraries: "OC Identifier tool"

In DNA microarray experiments, the gene fragments that are spotted on the slides are usually obtained by the synthesis of specific oligonucleotides that are able to amplify genes through PCR. Shotgun library sequences are an alternative to synthesis of primers for the study of each gene in the genome. The possibility of putting thousands of gene sequences into a single slide allows the use of shotgun clones in order to proceed with microarray analysis without a completely sequenced genome. We developed an OC Identifier tool (optimal clone identifier for genomic shotgun libraries) for the identification of unique genes in shotgun libraries based on a partially sequenced genome; this allows simultaneous use of clones in projects such as transcriptome and phylogeny studies, using comparative genomic hybridization and genome assembly. The OC Identifier tool allows comparative genome analysis, biological databases, query language in relational databases, and provides bioinformatics tools to identify clones that contain unique genes as alternatives to primer synthesis. The OC Identifier allows analysis of clones during the sequencing phase, making it possible to select genes of interest for construction of a DNA microarray.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers.

AIMS: Detection of Xylella fastidiosa in citrus plants and insect vectors. METHODS AND RESULTS: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay. CONCLUSIONS: The use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa.

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Comparison of the genomes of two Xanthomonas pathogens with differing host specificities.

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Caracterização de rizóbios (Bradyrhizobium japonicum) e produtividade da soja

A field study on the competion and on the dynamics of occupation of nodulation sites by naturalized and seed inoculated strains of Bradyrhizobium japonicum was conducted on a Dark Red Latosol. The experimental design consisted of randomized blocks, with eight replications of the following treatments: A) control without inoculation; B) control without inoculation, with nitrogen fertilizer; C) inoculation with about 107 rhizobia/g (8g/kg of seed); D) inoculation with about 1010 rhizobia/g (8g/kg of seed). The inoculant was prepared with the strains SMS-314 (= SEMIA-587) and SMS-463 (=SEMIA-5019 = 29W), officially recommended for inoculant production. The identification of the strains which formed nodules was made in samples collected at 10, 42 and 70 days after seed germination. The grains were harvested to evaluate production and nitrogen content. Sorological typification was made with antiserum prepared from antigens constituted by B. japonicum strains SEMIA-587, SEMIA-5019 and SEMIA-5052 (=USDA-6), using the immunodot method. The characterization of the strains in nodules revealed a small increase (6,2 to 8,7%) in the occurrence of nodules formed by the recommended strains for the three sampling dates. There was an expressive participation of the strain 5052 (serogroup USDA-6) in the formation of the nodules. It was also observed that the naturalized strains may contribute differently to the formation of the nodules during the cycle, depending on the time of sampling.

Fez-se um estudo de campo sobre a competição e a dinâmica de ocupação de sítios de nodulação de estirpes de Bradyrhizobium japonicum naturalizadas ou inoculadas em sementes. O ensaio foi instalado em Latossolo Vermelho Escuro utilizando blocos ao acaso, com 8 repetições dos seguintes tratamentos: A) controle sem inoculante; B) controle sem inoculante, com adubação nitrogenada; C) inoculante com cerca de 107 rizóbios/g (8g/kg de semente); D) inoculação com cerca de 1010 rizóbios/g (8g/kg de semente). Os inoculantes foram preparados com as estirpes recomendadas na ocasião, SMS - 314 (= SEMIA 587) e SMS - 463 (= SEMIA 5019 = 29W). A identificação das estirpes formadoras dos nódulos foi realizada em amostras coletadas aos 10, 42 e 70 dias após a germinação. Realizou-se também a colheita dos grãos para avaliação da produção e de N-total. A tipificação sorológica foi feita com antissoro preparado a partir de antígenos constituídos pelas seguintes estirpes de Bradyrhizobium japonicum: SEMIA 587, SEMIA 5019 e SEMIA 5052 (= USDA 6), utilizando-se da técnica "immunodot". A caracterização das estirpes dos nódulos revelou um pequeno aumento (6,2 a 8,7%), na ocorrência de nódulos formados pelas estirpes recomendadas, nas três épocas de amostragem. Houve expressiva participação da estirpe 5052 (= sorogrupo USDA 6) na formação dos nódulos. Verificou-se que as estirpes naturalizadas podem contribuir de forma diferente na formação dos nódulos durante o ciclo, dependendo da amostragem.

Caracterização de rizóbios (Bradyrhizobium japonicum) e produtividade da soja

A field study on the competion and on the dynamics of occupation of nodulation sites by naturalized and seed inoculated strains of Bradyrhizobium japonicum was conducted on a Dark Red Latosol. The experimental design consisted of randomized blocks, with eight replications of the following treatments: A) control without inoculation; B) control without inoculation, with nitrogen fertilizer; C) inoculation with about 107 rhizobia/g (8g/kg of seed); D) inoculation with about 1010 rhizobia/g (8g/kg of seed). The inoculant was prepared with the strains SMS-314 (= SEMIA-587) and SMS-463 (=SEMIA-5019 = 29W), officially recommended for inoculant production. The identification of the strains which formed nodules was made in samples collected at 10, 42 and 70 days after seed germination. The grains were harvested to evaluate production and nitrogen content. Sorological typification was made with antiserum prepared from antigens constituted by B. japonicum strains SEMIA-587, SEMIA-5019 and SEMIA-5052 (=USDA-6), using the immunodot method. The characterization of the strains in nodules revealed a small increase (6,2 to 8,7%) in the occurrence of nodules formed by the recommended strains for the three sampling dates. There was an expressive participation of the strain 5052 (serogroup USDA-6) in the formation of the nodules. It was also observed that the naturalized strains may contribute differently to the formation of the nodules during the cycle, depending on the time of sampling.

Fez-se um estudo de campo sobre a competição e a dinâmica de ocupação de sítios de nodulação de estirpes de Bradyrhizobium japonicum naturalizadas ou inoculadas em sementes. O ensaio foi instalado em Latossolo Vermelho Escuro utilizando blocos ao acaso, com 8 repetições dos seguintes tratamentos: A) controle sem inoculante; B) controle sem inoculante, com adubação nitrogenada; C) inoculante com cerca de 107 rizóbios/g (8g/kg de semente); D) inoculação com cerca de 1010 rizóbios/g (8g/kg de semente). Os inoculantes foram preparados com as estirpes recomendadas na ocasião, SMS - 314 (= SEMIA 587) e SMS - 463 (= SEMIA 5019 = 29W). A identificação das estirpes formadoras dos nódulos foi realizada em amostras coletadas aos 10, 42 e 70 dias após a germinação. Realizou-se também a colheita dos grãos para avaliação da produção e de N-total. A tipificação sorológica foi feita com antissoro preparado a partir de antígenos constituídos pelas seguintes estirpes de Bradyrhizobium japonicum: SEMIA 587, SEMIA 5019 e SEMIA 5052 (= USDA 6), utilizando-se da técnica "immunodot". A caracterização das estirpes dos nódulos revelou um pequeno aumento (6,2 a 8,7%), na ocorrência de nódulos formados pelas estirpes recomendadas, nas três épocas de amostragem. Houve expressiva participação da estirpe 5052 (= sorogrupo USDA 6) na formação dos nódulos. Verificou-se que as estirpes naturalizadas podem contribuir de forma diferente na formação dos nódulos durante o ciclo, dependendo da amostragem.