This thesis is a serological investigation of actinomycetes as a preliminary to taxonomic study.

This thesis is a spectrographic study of a select group of actinomycetes.

Orientadores: Fumio Yokota, Tasi Siu Mui / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T16:40:59Z (GMT). No. of bitstreams: 1 Garcia_CarlosEduardo_D.pdf: 3424911 bytes, checksum: 6338d2ede1846761521ec0cfb4c5e1ec (MD5) Previous issue date: 2006 / Doutorado / Doutor em Ciência de Alimentos

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.

Most microbial organic molecules are secondary metabolites which consist of diverse chemical structures and a range of biological activities. Actinobacteria form a large group of Eubacteria that are prolific producers of these metabolites. The recurrence of pathogens resistant to antibiotics and a wider use of these metabolites apart from their use as anti-infectives, has been the impetus for pharmaceutical companies to search for compounds produced by rare and existing actinobacterial cultures. Accessing microbial biosynthetic pathway diversity has been possible through the use of sensitive and innovative molecular detection methodologies. The present study evaluated the use of molecular based screening as a rational approach to detect secondary metabolite biosynthetic genes (SMBG) in uncharacterised natural Actinobacterial populations. A polymerase chain reaction (PCR) approach was selected for ease of application and high sample processivity. Rational designed screening approaches using PCR in the discovery of SMBG, involved identifying common functions in secondary metabolite biosynthetic pathways, such as condensation reactions in polyketide synthesis, genes encoding these functions, and using conserved regions of these genes as templates for the design of primers to detect similar sequences in uncharacterised actinobacteria. Design of primers involved rigorous in silico analysis followed by experimentation and validation. PCR screening was applied to 22 uncharacterised environmental isolates, eight of these displayed the presence of the ketosynthase (KS) gene belonging to the type I polyketide synthases and eight contained the ketosynthase (KSĄ) gene belonging to the type II polyketide synthases, six of the isolates contained the presence of a presumptive dTDP-glucose synthase (strD) gene which is involved in the formation of deoxysugar components of aminoglycoside antibiotics and one isolate contained the presence of a presumptive isopenicillin N synthase (pcbC) gene involved in beta-lactam synthesis. Alignments of partially sequenced PCR products from isolates A1488 and A3023 obtained using type II PKS primers showed close similarities with KSĄ genes from antibiotic producing actinobacteria. Similarly, alignments of sequences from isolates A1113 and A0350 showed regions of similarities to KS genes from antibiotic producing actinobacteria. Fermentation techniques were used for inducing expression of secondary metabolites from the uncharacterised actinobacteria isolates. By using antimicrobial guided screening it was determined that most of the isolates possessed the capacity to produce antimicrobial metabolites. Dominant antagonistic activity was detected against Gram positive bacteria and to a minor extent against fungi. Optimal fermentation liquid media were identified for certain isolates for the production of antimicrobial metabolites. Two alternative fermentation methods; solid-state and liquid-oil fermentations were evaluated to improve secondary metabolite production in the uncharacterised isolates. Solid-substrate fermentation showed that it could induce a complex metabolite pattern by TLC analysis, however this pattern varied according to the substrate being used. Liquid media supplemented with refined oils, showed a positive response indicated by higher antibacterial activities detected. Evaluation of semi-purified organic extracts identified two isolates A1113 and A0350 producing similar antimicrobial metabolites as detected by HPLC/UV/MS, a literature database search of similar compounds containing the same molecular weight identified the compound as belonging to the actinomycin group of compounds. A complex metabolic pattern was identified for isolate A2381, database searching identified some of the compounds as having similar molecular weights to actinopyrones, trichostatins, antibiotics PI 220, WP 3688-5 and YL 01869P. Drug discovery screening can serve to benefit from PCR detection of biochemical genotypes in initial screens, providing a rapid approach in identifying secondary metabolite producing capabilities of microorganisms prior to the commencement of costly and time consuming fermentation studies. Additionally the identification of biochemical genotypes allows a directed approach in using fermentation media designed to induce biosynthetic pathways of specific classes of compounds.

<p>The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats.</p>

New structurally diverse natural products can be discovered when carefully designed screening procedures have been applied and when a prolific organism from a different biological source is examined, such as, rare actinobacteria from an untapped environment. Chapter 3 describes the isolation and structure characterisation of eight compounds from the rare actinobacterum, Saccharothrix xinjiangensis (NRRL B-24321), including, two new 16-member macrolides, Tianchimycin A and B, respectively. OSMAC (One Strain - Many Compounds) is used to search bioactive compounds from the metabolic profile of S. xinjiangensis, isolated from a semi-arid or desert area, Tanchi, Xinjiang in the study. Isolated compounds were characterised by NMR spectroscopy and accurate mass spectrometric analysis. Investigations of the natural products at all levels, from genes, to enzymes, to molecules has revealed insights into differentiating features of the biosynthetic pathways that lead to structural diversity of natural products. The presence of a halogen substituent in natural products profoundly influences their biology activity. Actinomycins are a well-known class of antibiotics/anticancer agents. Here, the gene cluster directing chlorinated actinomycin G biosynthesis in Streptomyces iakyrus (DSM 41873) has been identified and sequenced. It contains one actinomycin synthetase I (ACMS I) gene and two copies of ACMS II and III genes. Genetic analysis demonstrates a unique partnership between the putative hydroxylation and chlorination activities as both acm8 and acm9 genes need to be transcribed for the biosynthesis of actinomycin G2 and actinomycin G3, respectively. In chapter 5, I descries a possible metabolic flux rebalancing pathway for increasing phenazinomycin production in S. iakyrus (DSM 41873) after interruption of the methyltrasfer gene (acmG5') in actinomycin G gene cluster. The gene cluster of phenazinomycin was identified by in silico analysis and by comparison with a known phenazine gene cluster from S. iakyrus (DSM 41873).

<p>The major aim of this study is to determine and compare the distribution of bacteria and actinobacteria in Marion Island terrestrial habitats.</p>

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.

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.