Metagenômica comparativa de solo de regiões de mata atlântica e caatinga do estado do Rio Grande do Norte - Brasil

The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored

Estudo do genoma do vírus causador da mionecrose infecciosa em camarões e desenvolvimento de métodos para detecção de diferentes variantes

Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites

Ecogenômica de archaea e monitoramento de comunidades de procariotos redutores de sulfato: aplicações na indústria de petróleo e gás

The human activities responsible for the ambient degradation in the modern world are diverse. The industrial activities are preponderant in the question of the impact consequences for brazilian ecosystems. Amongst the human activities, the petroliferous industry in operation in Potiguar Petroliferous Basin (PPB) displays the constant risk of ambient impacts in the integrant cities, not only for the human populations and the environment, but also it reaches the native microorganisms of Caatinga ground and in the mangrove sediment. Not hindering, the elaboration of strategies of bioremediation for impacted areas pass through the knowledge of microbiota and its relations with the environment. Moreover, in the microorganism groups associated to oil, are emphasized the sulfate-reducing prokaryotes (SRP) that, in its anaerobic metabolism, these organisms participate of the sulfate reduction, discharging H2S, causing ambient risks and causing the corrosion of surfaces, as pipelines and tanks, resulting in damages for the industry. Some ancestries of PRS integrate the Archaea domain, group of microorganisms whose sequenced genomes present predominance of extremophilic adaptations, including surrounding with oil presence. This work has two correlated objectives: i) the detection and monitoring of the gene dsrB, gift in sulfate-reducing prokaryotes, through DGGE analysis in samples of mDNA of a mangrove sediment and semiarid soil, both in the BPP; ii) to relate genomic characteristics to the ecological aspects of Archaea through in silico studies, standing out the importance to the oil and gas industry. The results of the first work suggest that the petrodegraders communities of SRP persist after the contamination with oil in mangrove sediment and in semiarid soil. Comparing the populations of both sites, it reveals that there are variations in the size and composition during one year of experiments. In the second work, functional and structural factors are the probable cause to the pressure in maintenance of the conservation of the sequences in the multiple copies of the 16S rDNA gene. Is verified also the discrepancy established between total content GC and content GC of the same gene. Such results relating ribosomal genes and the ambient factors are important for metagenomic evaluations using PCR-DGGE. The knowledge of microbiota associated to the oil can contribute for a better destination of resources by the petroliferous industry and the development of bioremediation strategies. Likewise, search to lead to the best agreement of the performance of native microbiota in biogeochemical cycles in Potiguar Petroliferous Basin ecosystem

Evolução do reparo por excisão de nucleotídeo

The phylogeny is one of the main activities of the modern taxonomists and a way to reconstruct the history of the life through comparative analysis of these sequences stored in their genomes aimed find any justification for the origin or evolution of them. Among the sequences with a high level of conservation are the genes of repair because it is important for the conservation and maintenance of genetic stability. Hence, variations in repair genes, as the genes of the nucleotide excision repair (NER), may indicate a possible gene transfer between species. This study aimed to examine the evolutionary history of the components of the NER. For this, sequences of UVRA, UVRB, UVRC and XPB were obtained from GenBank by Blast-p, considering 10-15 as cutoff to create a database. Phylogenetic studies were done using algorithms in PAUP programs, BAYES and PHYLIP package. Phylogenetic trees were build with protein sequences and with sequences of 16S ribosomal RNA for comparative analysis by the methods of parsimony, likelihood and Bayesian. The XPB tree shows that archaeal´s XPB helicases are similar to eukaryotic helicases. According to this data, we infer that the eukaryote nucleotide excision repair system had appeared in Archaea. At UVRA, UVRB and UVRC trees was found a monophyletic group formed by three species of epsilonproteobacterias class, three species of mollicutes class and archaeabacterias of Methanobacteria and Methanococci classes. This information is supported by a tree obtained with the proteins, UVRA, UVRB and UVRC concatenated. Thus, although there are arguments in the literature defending the horizontal transfer of the system uvrABC of bacteria to archaeabacterias, the analysis made in this study suggests that occurred a vertical transfer, from archaeabacteria, of both the NER genes: uvrABC and XPs. According the parsimony, this is the best way because of the occurrence of monophyletic groups, the time of divergence of classes and number of archaeabacterias species with uvrABC system

Prospecção de genes de interesse biotecnológico : uma abordagem metagenômica

The total number of prokaryotic cells on Earth has been estimated at 4 to 6x1030 and only about 1% of microorganisms present in the environment can be cultivated by standard techniques of cultivation and plating. Therefore, it is a huge biological and genetic pool that can be exploited, for the identification and characterization of genes with biotechnological potential. Within this perspective, the metagenomics approach was applied in this work. Functional screening methods were performed aiming to identify new genes related to DNA repair and / or oxidative stress resistance, hydrocarbon degradation and hydrolytic activities (lipase, amylase and protease). Metagenomic libraries were built utilizing DNA extracted from soil samples collected in João Câmara RN. The libraries were analyzed functionally using specific substrate containing solid medium (hydrolytic activity), supplemented with H2O2 (DNA repair and / or resistance to oxidative stress) and liquid medium supplemented with light Arabian oil (activity, degradation of hydrocarbons). After confirmation of activity and exclusion of false-positive results, 49 clones were obtained, being 2 positive for amylase activity, 22 resistant to oxidative stress generated by H2O2 and 25 clones active for hydrocarbons degradation. Analysis of the sequences showed hypothetical proteins, dienelactona hydrolase, DNA polymerase, acetyltransferase, phosphotransferase, methyltransferase, endonucleases, among other proteins. The sequence data obtained matched with the functions tested, highlighting the success of metagenomics approaches combined with functional screening methods, leading to very promising results

Estudos ecogenômicos e bioprospectivos de Shewanella ssp

Bacteria trom Shewanella and Geobacter ganera are the most studied iron-reducing microorganisms particularly due to their electron transport systems and contribution to some industrial and environmental problems, including steel corrosion, bioenergy and bioremediation of petroleum-impacted sites. The present study was focused in two ways: the first is an in silico comparative ecogenomic study of Shewanella spp. with sequenced genomes, and the second is an experimental metagenomic work to detect iron-reducing Shewanella through PCR-DGGE of a metabolic gene. The in silico study resulted in positive correIation between copy number of 16S rDNA and genome size in Shewanella spp., with clusters of rrn near lhe origin of replication. This way, the genus is inferred as opportunist. There are no compact genomes and their sequences length varied, ranging from 4306142 nt in S. amazonensis SB2B to 5935403 nt in S. woodyi ATCC 51908, without correIation to temperature range characteristic of each specie. Intragenomic 16S rDNA sequences possess little divergence, but reasonable to resuIt in different phyIogenetic trees, depending on the sequence that is chosen to compare. For moIecuIar detection of iron-reducing Shewanella, it is proposed the mtrB gene as new biomarker. because it codes to a fundamental protein at Fe (III)-reduction. The specific primers were designed and evaluated in silico and resulted in a fragment of 360 pb. In the second study, these primers were tested in a genomic sample from S. oneidensis MR-1, amplifying the expected region. After this successfuI resuIt, the primer set was used as a tool to assess the iron-reducing communities of ShewaneIla genus under an environmental stress, i.e. crude oil contamination in mangrove sediment in Rio Grande do Norte State (Brazil). The primers presented high specificity and the reactions performed resulted in one single band of ampIification in the metagenomic samples. The fingerprinting obtained at DGGE reveaIed temporal variation of Shewanella spp. in analyzed samples. The resuIts presented show the detection of a biotechnological important group of microorganisms, the iron-reducing Shewanella spp. using a metabolic gane as target. It is concluded there are eight or more 16S rDNA sequences in Shewanella genus, with little divergence among them that affects the phylogeny; the pair of primers designed to ampIify mtrB sequences is a viable alternative to detect iron-reducing ShewanelIa in metagenomic approaches; such bacteria are present in the mangrove sediment anaIyzed, with temporal variations in the samples. This is the first experimental study that screened the iron-reducing Shewanella genus in a metagenomic experiment of mangrove sediments subjected to oil contamination through a key metabolic gene