Detection of Genes that Encode Naphthalene Dioxygenase, Toluene Dioxygenase and Alkane Hydroxylase of Petroleum-degrading Bacteria

Foram avaliados 14 isolados degradadores de petróleo, quanto à diversidade genética e à atividade catabólica dos mesmos e de cinco destes, em consórcio, no meio de cultivo adicionado de petróleo por meio da técnica de RT-PCR. A diversidade genética dos 14 isolados bacterianos foi avaliada por RAPD utilizando 12 oligonucleotídeos. O padrão de bandas amplificado do DNA total dos isolados evidenciou uma alta diversidade genética entre os isolados LBBMA 1, LBBMA 18a, LBBMA 53, LBBMA 88b, LBBMA 161, LBBMA 191, LBBMA 201 e LBBMA ES11, quando comparado ao padrão de bandas obtidos com os isolados LBBMA 58, LBBMA 75, LBBMA 101b, LBBMA 105a, LBBMA 195 e LBBMA 199. A detecção de seqüências genômicas e dos respectivos transcritos dos genes que codificam alcano hidroxilase, naftaleno dioxigenase e tolueno dioxigenase em meio de cultivo adicionado de petróleo evidenciou que os isolados LBBMA 58, LBBMA 75, LBBMA 101b e LBBMA 199 foram promissores para serem utilizados no processo de biorremediação. O consórcio C7 (LBBMA 105a, LBBMA 191, LBBMA 195, LBBMA 199, LBBMA 201) foi selecionado para ser avaliado quanto à atividade catabólica em meio de cultivo adicionado de petróleo, nos tempos: 0,5 hora, 4 horas, 7 horas e viii24 horas. Detectou-se neste consórcio, os transcritos correspondentes aos genes que codificam alcano hidroxilase e naftaleno dioxigenase em todos os tempos avaliados. Estes resultados mostraram que a avaliação da atividade catabólica por meio da detecção dos transcritos indicou o potencial de aplicação deste consórcio no processo de biorremediação.The present study objectified to evaluate the genetic diversity among 14 Petroleum-degrading bacterial isolates and to verify the catabolic activity of each one separately and in consortium in culture medium with addition of petroleum by RT-PCR technique. Their genetic diversity was evaluated by RAPD using twelve primers. The isolates presented high polymorphism indicating a great genetic diversity among them. The evaluation of the potential and the catabolic activity of the isolates was accomplished using as markers three catabolic genes that encode alkane hydroxylase, naphthalene dioxygenase and toluene dioxygenase. In these isolates different results were observed for the presence of the genomic sequences and their respective transcripts. These results indicated that the isolates LBBMA 58, LBBMA 75, LBBMA 101b and LBBMA 199 are promising for the bioremediation process. The consortium C7 (LBBMA 105a, LBBMA 191, LBBMA 195, LBBMA 199, LBBMA 201) was chosen to have its catabolic activity appraised due to its greater efficiency. In this consortium transcripts corresponding to the genes that encode alkane hydroxylase and naphthalene dioxygenase were detected with 0,5 hour, 4 xhours, 7 hours and 24 hours. These results indicated the potential of this consortium for bioremediation.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Prospection of phenol-degrading activity in microbial metagenome from petroleum refinery wastewater

Orientador: Valéria Maia MerzelTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os efluentes de refinarias contêm vários compostos tóxicos, que podem provocar sérios danos aos seres vivos se não tratados previamente. Os fenóis são poluentes encontrados em efluentes de diversas indústrias, incluindo as refinarias de petróleo, e são conhecidos como um dos compostos mais tóxicos encontrados no meio ambiente. Portanto, há necessidade constante de buscar formas de remover ou reduzir a presença destas substâncias nos efluentes. Este trabalho teve como objetivos analisar e explorar a diversidade microbiana presente em lodo de sistemas de tratamento de efluentes de refinarias petróleo, através da construção de bibliotecas de genes RNAr 16S e bibliotecas metagenômicas em vetores do tipo fosmídeo. Análises filogenéticas das bibliotecas de genes RNAr 16S e metagenômicas mostraram uma comunidade bacteriana bastante diversa, com predominância do Filo Proteobacteria, seguido de Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi e Bacteroidetes. Dentre os gêneros mais abundantes, se destacaram Thauera, Comamonas, Diaphorobacter e Thiobacillus. Espécies pertencentes a estes gêneros realizam o processo de desnitrificação e espécies de Thauera e Comamonas estão relacionadas com a degradação de compostos aromáticos. A grande diversidade bacteriana refletiu em uma ampla diversidade metabólica, sendo obtido um grande número de resultados para genes relacionados com processos biológicos importantes para o tratamento de efluentes, como o metabolismo de nitrogênio, enxofre, fósforo, além de genes relacionados com o catabolismo de compostos aromáticos, tais como benzoato, bifenil e fenol. Adicionalmente, foram observadas possíveis novas sequências para os genes que codificam enzimas-chaves responsáveis pela meta-clivagem de fenol e outros aromáticos, como fenol hidroxilase e catecol 2,3-dioxigenase. No presente estudo foram também realizadas triagens funcionais dos 13.200 clones para enzimas hidrolíticas, com detecção de dois hits para esterase e um para lipase. Este trabalho mostrou que a bioprospecção genômica microbiana é uma estratégia inovadora na área de tratamento de efluentes, que pode contribuir para o conhecimento da microbiota responsável pela degradação dos poluentes.Abstract: Refinery wastewater has several toxic compounds that may cause serious damage to life. Phenolic compounds are found in several industrial effluents, including petroleum refinery, and represent a significant environmental toxicity hazard. Therefore, there is a constant need to search for new technologies able to remove and reduce the presence of these substances in wastewater. This study aimed to analyze and explore the microbial diversity present in sludge from refinery wastewater treatment systems through the construction of 16S rRNA genes libraries and metagenomic libraries in fosmid vectors. Sequencing and phylogenetic analyses of libraries showed a highly diverse bacterial community, with predominance of the Proteobacteria phylum, followed by Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi and Bacteroidetes. The most abundant genera were Thauera, Comamonas, Diaphorobacter and Thiobacillus. Species belonging to these genera are facultative anaerobic and may be involved with the denitrification process, and Thauera and Comamonas species are related with degradation of aromatic compounds. This bacterial diversity reflected in a broad metabolic diversity. Analysis of the metagenomic data derived from pyrosequencing revealed a lot of hits related with biological processes of great relevance for wastewater treatment, such as genes for nitrogen, sulfur and phosphorus metabolism, in addition to genes related to the catabolism of aromatic compounds, such as benzoate, biphenyl and phenol. Additionally, sequences representing possible new genes encoding for key-enzymes responsible for the meta-cleavage of phenol and other aromatic compounds, such as phenol hydroxylase and cathecol 2,3-dioxygenase, were found in the fosmid libraries. Finally, high-throughput functional screening of 13,200 clones for hydrolytic enzymes yielded two positive hits for lipase and one for esterase. The data gathered together in this work showed that microbial genomic prospection is an innovative strategy for the wastewater treatment system that may undoubtedly contribute to the knowledge of microorganisms responsible by pollutant degradation.DoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

Farm characteristics.

<p>Collected from interviews.</p><p>^a Distance between each coffee tree in a row versus distance between rows (in meter).</p><p>Farm characteristics.</p

LEfSe discriminatory OTUs among coffee rhizosphere samples.

<p>Heatmap of LEfSe results. The number of discriminatory OTUs in each phylum is shown in corresponding box for each of the three sample sites (INT, TRN, and ORG). Statistic calculated in mothur and results plotted in R.</p

Most prevalent phyla.

<p>Taxonomic classification raking the 1^st, 2^nd, and 3^rd most prevalent phylum in each of the six sample sites. Data were classified using mothur’s classify.seqs command and GreenGenes as the reference database (May 2013). In parentheses is the relative abundance of each phylum as a percentage of the total number of sequences within each site.</p><p>Most prevalent phyla.</p

Summary data of OTUs, richness, and diversity.

<p>Values shown for each of the six sites (INT, TRN, or ORG) and each sample type (coffee or control) combinations, calculated using mothur. OTUs were clustered at 97% sequence similarity.</p><p>^a CI = confidence interval</p><p>Summary data of OTUs, richness, and diversity.</p

Chao richness estimates of species-level OTUs.

<p><b>(A)</b> Richness level calculated for each of 81 samples, color-coded and grouped by site (INT, TRN, or ORG) and sample type (coffee rhizosphere or soil control) as detailed in the methods. Richness estimates (± 95% confidence intervals) of 97% similarity OTUs calculated in mothur and plotted in R. Black circle indicates number of observed OTUs. <b>(B)</b> Mean Chao richness estimates (± standard error) from individual samples shown in Fig 1A within each sample site and sample type calculated in R.</p

16S rRNA Sequence Data, Brazilian Coffee Soils

<p>16S sequencing data for DNA extracted from soils from Brazillian coffee farms. Sequenced on Illumina MiSeq with primers from Caporaso (2011, 2012).</p

Conscientização dos produtores de leite da Zona da Mata Mineira sobre métodos de prevenção da mastite bovina e isolamento dos seus agentes etiológicos

Erro no DOI, por isso foi disponibilizado o link de acesso.A mastite causa perda da qualidade do leite e riscos à saúde dos consumidores em virtude da presença de resíduos de antibióticos e microrganismos patogênicos, além de sérios prejuízos econômicos tanto para o produtor quanto para a indústria de laticínios. O conhecimento referente aos agentes causadores da doença, à sua evolução clínica e a fatores relacionados com o estabelecimento da infecção deve ser colocado em prática em programas de controle e prevenção da doença, visando o treinamento dos produtores, principalmente em respeito ao manejo adequado de ordenha. Neste trabalho, buscamos conscientizar produtores de leite da Zona da Mata mineira sobre a importância do controle e prevenção de mastite bovina e determinar seus principais agentes etiológicos. Identificamos 3,3% de casos de mastite clínica, 15% de subclínica e 81,7% de tetos sadios em vacas leiteiras da região. Como principais agentes etiológicos, foram isolados o Staphylococcus aureus em 28,52% dos casos e o Staphylococcus sp., coagulase negativa, em 14,83%. Observamos também que poucos produtores da região possuem conhecimentos básicos sobre os métodos de prevenção e controle da mastite. Sendo assim, muitos carecem de programas de assistência técnica especializada, o que seria útil para o desenvolvimento de suas comunidades.Mastitis causes loss of milk quality, health risks to consumers due to antibiotic residues and pathogenic microbes, and serious economic losses to both the grower and the dairy industry. The knowledge related to the causative species of the disease, clinical evolution and factors that favour the establishment of the infection should be prioritized. Improved control programs and disease prevention should be put into practice, aiming the suitable training of the producers, mainly referring to the proper handling of milking. In this work, we seek to carry out an awareness of the importance of bovine mastitis control and prevention in the Zona da Mata mineira and to determine the main etiological agents. We identified 3.3% of cases of clinical mastitis, 15% subclinical and 81.7% of ceiling healthy. The main etiologic agents were Staphylococcus aureus isolated in 28.52% of cases and Saphylococcus sp., coagulase negative, 14.83%. We also observed that few producers in the region have basic knowledge about methods of mastitis prevention and control. Thus, many programs lack the specialized technical assistance, which would be useful for the development of their communities

Screening and characterization of prophages in Desulfovibrio genomes

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies