No description available.

No description available.

The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.

As interações que ocorrem entre as bactérias e amebas podem dar-se através de relações mútuas, onde ambos os organismos se beneficiam da associação ou parasitárias em que um organismo se beneficia em detrimento do outro. A convivência de vários microrganismos que compartilham o mesmo ambiente pode produzir alterações seja no crescimento dos organismos, nos padrões de adaptação, na morfologia, no seu desenvolvimento, ou até mesmo na sua capacidade para sintetizar proteínas e metabólitos secundários. Neste estudo, é avaliada a interação entre Acanthamoeba polyphaga e Streptomyces sp. através de cocultivo, com objetivo de obter extratos brutos com ação antimicrobiana. No cocultivo, as amebas inviabilizaram na presença da bactéria. Após contato com as amebas houve alteração morfológica em Streptomyces sp. em todos os tempos de incubação, com produção de hifas, diferente do controle que permaneceu na fase de esporos. A partir do cocultivo foi possível obter extrato bruto em 50 dias, sendo avaliados em diferentes tempos de incubação (1º, 7º, 14º, 21º e 28º dias), contra bactérias multirresistentes como Escherichia coli e Pseudomonas aeruginosa, mostrando atividade antimicrobiana, tanto no cocultivo quanto no controle. Com análise estatística foi possível verificar que os extratos produzidos em 24 horas (1º) apresentaram maior atividade, especialmente contra P. aeruginosa. Os extratos produzidos pelo cocultivo e controle se comportaram diferentemente um do outro, porém as diferenças não foram estatisticamente significativas. Em relação à biomassa produzida, foi observado maior volume de biomassa no cocultivo, do que no controle, indicando que o contato entre os dois microrganismos favoreceu a produção de massa celular, porém não houve diferença significativa, somente quando comparado entre dias. Estes resultados mostram que há interação entre Acanthamoeba e Streptomyces uma vez que, a bactéria se beneficiou da ameba auxiliando no seu desenvolvimento. Esta interação entre os microrganismos pode ser importante na modulação da produção de substâncias de ação antimicrobiana, fato que ainda necessita investigação. / The interactions that occur between bacteria and amoebas can give through mutual relations, where both organisms benefit from the association or parasitic in which one organism benefits at the expense of the other. The coexistence of various microorganisms share the same environment can produce alterations in the growth of the organisms is, patterns of adaptation in morphology, development, or even in their ability to synthesize proteins and secondary metabolites. This study evaluates the interaction between Acanthamoeba polyphaga and Streptomyces sp. through cocultivation, in order to obtain crude extracts with antimicrobial action. In cocultivation, amoebas made it impossible in the presence of the bacteria. After contact with amoebae were morphological changes in Streptomyces sp. All incubation times with hyphae production, different control spores this remained in phase. From the cocultivation it was possible to obtain crude extract in 50 days, being evaluated in different incubation times (1º, 7º, 14º, 21º and 28º days), against multiresistant bacteria such as Escherichia coli and Pseudomonas aeruginosa, showing antimicrobial activity in both the cocultivation and in control. With statistical analysis found that the extracts in 24 hours (1º) showed greater activity, especially against P. aeruginosa. The extracts produced by the coculture and control behaved differently from one another, but the differences were not statistically significant. Regarding the biomass produced, there was a higher volume of biomass in cocultivation, than in the control, indicating that the contact between the two organisms favored the cell mass production, but there was no significant difference only when compared between days. These results show that there is interaction between Acanthamoeba and Streptomyces since the bacteria benefited amoeba assisting in their development. This interaction between microorganisms may be important in modulating the production of antimicrobial substances, a fact that still requires investigation.

Graduation date: 1999

Graduation date: 1996

Graduation date: 1996

No description available.

Plant biomass serves as a carbon and energy source for Streptomyces spp. which secrete degradative enzymes capable of breaking down the complex plant biomass into simple saccharides. Hemicellulose is a major component of plants and is composed of five and six carbon sugars, such as xylose and glucose. Enzymatic degradation of hemicellulose to obtain desired sugars has been a cornerstone of many industries, as well as the subject of worldwide research for additional sources of efficient enzymes for substrate conversion. In this study, environmentally-derived Streptomyces isolates were screened for their ability to hydrolyze oat-spelt and birchwood xylan in agar-based high throughput activity screens. Of the isolates tested, eight displayed high levels of substrate-degrading activity and were chosen for further characterization which included 16S rRNA gene analysis, microscopic analysis from both liquid and agar grown cultures, xylanase-specific activity, lignin peroxidase production and indole acetic acid production.Qualitative assessment of extracellular lactone signalling for all eight isolates was also performed. Putative lactone signalling was observed for Streptomyces isolates JLS1-C4, JLS1-A6, JLS2-D6 and KT1-B1 which exhibited xylanase-specific activities of 0.622 μmol/min/mg, 0.0243 μmol/min/mg, 0.721 μmol/min/mg, and 0.706 μmol/min/mg respectively. Streptomyces isolates JLS1-F12 and JLS1-C12 did not exhibit lactone signalling but did exhibit xylanase-specific activities of 0.125 μmol/min/mg and 0.0688 μmol/min/mg respectively. No xylanase-specific activity was detected for isolates JLS2- C7 and KT1-B8; however lactone signalling was observed for isolate KT1-B8. Streptomyces isolate JLS1-A6 degraded birchwood xylan optimally at pH 4 and 28°C with a maximal xylanase activity of 1.56 x10-3 μmol/min/mg. / UOIT

Graduation date: 1995