Análise proteômica diferencial de Herbaspirillum seropedicae

Orientador: Luciano Fernandes HuergoCoorientadora: Ana Claudia BonattoMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências BiológicasResumo : Herbaspirillum seropedicae é uma bactéria diazotrófica endofítica encontrada colonizando plantas como milho, trigo, arroz e cana-de-açúcar. O objetivo deste trabalho foi determinar a ocorrência de possíveis modificações pós-traducionais e variações de expressão da enzima glutamina sintetase de H. seropedicae em resposta aos níveis de amônio no meio de cultivo. Nos experimentos realizados, as estirpes SMR1, LNglnKdel(glnK- ) e LNglnB(glnB- ) foram cultivadas em meio NFbHPmalato contendo 2mmol/l ou 20mmol/l de NH4Cl, como fonte de nitrogênio. Após o cultivo, as células foram coletadas, rompidas por sonicação e as proteínas solúveis foram fracionadas por eletroforese bidimensional. Os géis foram corados com Coomasie Blue Coloidal, as imagens foram digitalizadas e analisadas pelo programa ImageMaster 2D Platinum v.6.01. Três bandas com aproximadamente a mesma massa molecular, mas com pI diferentes foram retirados dos géis das estirpes estudadas e digeridas com tripsina, e os peptídeos obtidos analisados por um Espectrômetro de Massas do tipo MALDI-ToF/ToF. A análise revelou que essas bandas correspondiam a isoformas da enzima glutamina sintetase, o que foi confirmado por imunodetecção com anticorpos anti-GS, revelando quatro bandas com aproximadamente mesma massa, mas com pI diferente. A análise da variação de volume das isoformas da GS entre a estirpe selvagem e os mutantes revelou que as isoformas adenililada e desadenililada apresentaram padrão de variação (% volume) diferente do selvagem na estirpe mutante LNglnKdel(glnK- ). Já a nova isoforma de GS, com pI mais ácido, apresentou padrão de variação diferente em relação ao selvagem na estirpe mutante LNglnB(glnB- ). Esses resultados indicam que as proteínas PII participam da regulação da proteína glutamina sintetase em H. seropedicae. Além disso, a presença de duas outras isoformas da enzima glutamina sintetase, além das isoformas adenililada e desadenililada, sugere que o controle fino da atividade dessa enzima é de fundamental importância para o metabolismo nitrogenado e energético de Herbaspirillum seropedicae

Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO + H as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H) and the other steel mill off-gas (2% H). Results were characterized using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimization of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens

Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum

Introduction quantification of tetrahydrofolates (THFs), important metabolites in the wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxyge

Functional Categorization of Transcriptome in the Species <i>Symphysodon aequifasciatus</i> Pellegrin 1904 (Perciformes: Cichlidae) Exposed to Benzo[a]pyrene and Phenanthrene

<div><p>This study aims to evaluate the transcriptome alterations, through cDNA libraries, associated with the combined effects of two PAHs, benzo[a]pyrene (0.5 µg/L) and phenanthrene (50 µg/L), present in crude oil, on specimens of <i>Symphysodon aequifasciatus</i> (discus fish) after 48 h of exposure. The cDNA libraries were constructed according to the SOLiD™ SAGE™ protocol for sequencing in the SOLiD v.3 Plus sequencer. The results were analyzed by bioinformatics and differentially expressed genes were categorized using the gene ontology program. The functional categories (terms) found in the gene ontology and the gene network generated using STRING software were used to predict the adverse effects of benzo[a]pyrene and phenanthrene in the liver. In the present study, 27,127 genes (compared to <i>Danio rerio</i> database) were identified. Considering only those genes with a p-value less than or equal to 0.05 and greater than or equal to two-fold change in expression across libraries, we found 804 genes, 438 down-regulated (54%) and 366 up-regulated (46%), in the experimental group compared to the control. Out of this total, 327 genes were successfully categorized, 174 down-regulated and 153 up-regulated, using gene ontology. Using String, the gene network was composed by 199 nodes, 124 of them resulting in 274 interactions. The results showed that even an acute exposure of 48 h caused metabolic change in response to environmental contaminants, resulting in changes of cell integrity, in oxidation-reduction processes, in the immune response and disturbances of intracellular signaling of discus fish. Also the gene network has showed no central interplay cluster, exhibiting instead interconnected clusters interactions and connected sub-networks. These findings highlight that even an acute sublethal exposure of PAHs can cause metabolism changes that may affect survival of discus. Our findings using SOLiD coupled with SAGE-method resulted in a powerful and reliable means for gene expression analysis in discus, a non-model Amazonian fish.</p></div

Comparison of Top20 GO terms found in the Cellular Component category.

<p>Percentage of Top20 terms related to the down-regulated (black bars) and up-regulated genes (white bars), selected in the Cellular Component category.</p