Submitted by Alisson Mota (alisson.davidbeckam@gmail.com) on 2015-07-06T20:07:08Z No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:34:18Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:36:21Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-07-06T20:40:04Z (GMT) No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) / Made available in DSpace on 2015-07-06T20:40:04Z (GMT). No. of bitstreams: 1 Dissertação - Ingrid Reis da Silva.pdf: 9439671 bytes, checksum: 3eaa62300bf402ca99f08e88685a0dcb (MD5) Previous issue date: 2012-02-24 / Não Informada / The increasing number of antibiotic-resistant bacteria encourages the search for new antibacterial substances. Therefore, the selection of microorganisms with potential for production of new antimicrobial compounds have been extensively studied. Among these organisms a special attention is given to the actinomycetes that have the capacity to produce a variety of bioactive compounds such as antibiotics, antifungal, antitumor and other compounds that can be applied in various industry segments. The genus Streptomyces is considered of great industrial importance due to its ability to produce many secondary metabolites, accounting for 80% of currently used antibiotics. Considering the importance of actinomycetes and existing biodiversity in the Amazon, this study aims to isolate and select actinomycetes producing antibiotics and optimize their production. In this sense, he was made an initial screening to detect the antimicrobial activity of 371 actinomycetes isolated from soil from different localities in the Amazon region. Antibiosis trials were conducted to evaluate the antimicrobial activity against the indicator microorganisms isolated Gram-positive and Gram-negative. From these preliminary results, three isolates were considered promising because it showed inhibitory activity against Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 292123. These were selected for the study of production, using the response surface model to assess the best physical and chemical conditions that might interfere with production of the antibiotic of interest. The results presented here demonstrate that the isolate No. 01 is a potential producer of new bioactive metabolites. Morphological characterization and partial sequence analysis of 16S rDNA, demonstrate the great diversity of this group of microorganisms, and can thus identify the genus level. It has been shown that environmental conditions and the substrate are critical in the production of secondary metabolites, especially antibiotics. / O aumento crescente de bactérias resistentes a antibióticos incentiva à pesquisa por novas substâncias antibacterianas. Diante disso, a seleção de microrganismos com potencial para a produção de novos compostos antimicrobianos tem sido amplamente estudada. Dentre estes microrganismos uma especial atenção é dada aos actinomicetos que apresentam capacidade de produzir uma variedade de compostos bioativos como antibióticos, antifúngicos, antitumorais entre outros compostos que podem ser aplicados nos mais diversos segmentos da indústria. O gênero Streptomyces é considerado de grande importância industrial devido à sua capacidade de produzir muitos metabólitos secundários, respondendo por 80% dos antibióticos utilizados atualmente. Considerando a importância dos actinomicetos e a biodiversidade existente na Amazônia, este trabalho tem como objetivo isolar e selecionar actinomicetos produtores de antibióticos e otimizar a produção dos mesmos. Neste sentido, foi feito uma triagem inicial para detectar a atividade antimicrobiana dos 371 actinomicetos isolados de solo de diferentes localidades da região Amazônica. Foram realizados ensaios de antibiose para avaliar a atividade antimicrobiana dos isolados frente aos microrganismos indicadores Gram-positivos e Gram-negativos. A partir desses resultados preliminares, 3 isolados foram considerados promissores, pois apresentaram atividade inibitória frente a Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 e Enterococcus faecalis ATCC 292123. Estes,foram selecionados para o estudos de produção, utilizando o modelo de superfície de resposta, para avaliar as melhores condições físicas e químicas que possam interferir na produção do antibiótico de interesse. Os resultados apresentados neste trabalho demonstraram que o isolado n° 01 é um potencial produtor de novos metabólitos bioativos. A caracterização morfológica e a análise da seqüência parcial da região 16S do rDNA, demonstram a grande diversidade deste grupo de microrganismos, sendo possível assim, a identificação a nível de gênero. Foi demonstrado que as condições ambientais e do substrato são fundamentais na produção de metabólitos secundários, principalmente antimicrobianos.

Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:21:58Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:22:46Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-04-20T13:23:00Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) / Made available in DSpace on 2017-04-20T13:23:00Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese - Ingrid R. Silva.pdf: 12430919 bytes, checksum: 9f3d03c00cee59539e4bf02922a71940 (MD5) Previous issue date: 2016-12-16 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / The increase of antibiotic-resistant bacteria encourages the search for new antibacterial substances. Therefore, the selection of microorganisms with potential for the production of new antimicrobial compounds has been extensively studied. Among these microorganisms, special attention is given to actinomycetes that are capable of producing a wide variety of bioactive compounds, such as antibiotics, antifungals, antitumorals and other compounds that can be applied in various segments of the industry. The genus Streptomyces is considered of great industrial importance due to its capacity to produce many secondary metabolites, accounting for 80% of the antibiotics currently used. In previous studies, it was possible to report the antimicrobial activity of a strain of Streptomyces spp. Which showed inhibitory activity against Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 292123. Subsequently, its tuberculicidal activity was also inhibited by the growth of M. tuberculosis H37Rv (ATCC 27294). From these preliminary results, the present work had the objective of analyzing extracts and fractions of metabolites of Streptomyces sp. Aiming to isolate, purify and chemically identify the compound (s) with antimicrobial and tuberculicidal activity. The fractions in hexane (fr-Hex), ethyl acetate (fr-AcoEt) and chloroform (fr-Clo) were obtained from the metabolites obtained in liquid AC medium and by liquid-liquid partition using solvents of increasing polarity. The fr-Clo was chosen to continue the tests because its yield was higher, presenting high antibacterial activity in the Minimum Inhibitory Concentration (MIC) tests against the bacteria Staphylococcus aureus and Mycobacterium smegmatis. Thus, the fr-Clo was subjected to thin layer chromatography (CCD) using several eluent systems and then the Bioautography technique was performed to identify the compounds with activity. From this purification two bioactive sub-fractions were obtained, which were subjected to the isolation of the compounds by preparative CCD and HPLC-DAD-MS technique. The isolated substances were named Clarin A and Clarin B, which presented a Minimum Inhibitory Concentration (MIC) of 1.0 μg / mL for Clarin A and 0.5 μg / mL for Clarin B against Staphylococcus aureus and 16 μg / Ml against Mycobacterium smegmatis. They did not present cytotoxicity to 3T3-L1 non-tumor cells. The Clarin A and Clarin B substances were analyzed using Nuclear Magnetic Resonance (NMR) and low resolution and high resolution Mass Spectrometry techniques for chemical identification. The isolated substances were identified as Actinomycin D and Actinomycin X2 with m / z of 1277 [M + Na] and 1291 [M + Na]. The strong antibacterial activity of the isolated substances makes this product an important source of natural antibacterial compounds. / O aumento de bactérias resistentes a antibióticos incentiva a pesquisa por novas substâncias antibacterianas. Diante disso, a seleção de microrganismos com potencial para a produção de novos compostos antimicrobianos tem sido amplamente estudada. Dentre estes microrganismos uma especial atenção é dada aos actinomicetos que apresentam capacidade de produzir uma ampla variedade de compostos bioativos como antibióticos, antifúngicos, antitumorais entre outros compostos que podem ser aplicados nos mais diversos segmentos da indústria. O gênero Streptomyces é considerado de grande importância industrial devido à sua capacidade de produzir muitos metabólitos secundários, respondendo por 80% dos antibióticos utilizados atualmente. Em trabalhos anteriores, foi possível reportar a atividade antimicrobiana de uma linhagem de Streptomyces spp. que apresentou atividade inibitória frente a Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 e Enterococcus faecalis ATCC 292123. Posteriormente, verificou-se ainda a sua atividade tuberculicida inibindo o crescimento da M. tuberculosis H37Rv (ATCC 27294). A partir desses resultados preliminares, o presente trabalho teve por objetivo analisar os extratos e frações de metabólitos de Streptomyces sp. visando isolar, purificar e identificar quimicamente o(s) composto(s) com atividade antimicrobiana e tuberculicida. A partir dos metabólitos obtidos em meio AC líquido e por partição líquido-líquido utilizando solventes de polaridade crescente, obteve-se as frações em hexano (fr-Hex), acetato de etila (fr-AcoEt) e clorofórmio (fr-Clo). A fr-Clo foi escolhida para dar continuidade aos ensaios pois o seu rendimento foi maior, apresentando alta atividade antibacteriana nos testes de Concentração Inibitória Mínima (CIM), contra as bactérias Staphylococcus aureus e Mycobacterium smegmatis. Dessa forma, a fr-Clo foi submetida à Cromatografia em Camada Delgada (CCD) utilizando vários sistemas de eluentes e, em seguida, a técnica de Bioautografia foi realizada para identificar os compostos com atividade. Dessa purificação foram obtidas duas subfrações bioativas, as quais foram submetidas ao isolamento dos compostos pela técnica de CCD preparativa e HPLC – DAD-MS. As substâncias isoladas foram denominados Clarina A e Clarina B, as mesmas apresentaram uma Concentração Inibitória Mínima (CIM) de 1,0 μg/mL para a Clarina A e 0,5 μg/mL para Clarina B frente a bactéria Staphylococcus aureus e 16 μg/mL frente a Mycobacterium smegmatis. As mesmas não apresentaram citotoxidade para céluas não tumorais 3T3-L1. As substâncias Clarina A e Clarina B foram analisadas por meio das técnicas de Ressonância Magnética Nuclear (RMN) e Espectrometria de Massas de baixa e alta resolução para identificação química. As substâncias isoladas foram identificadas como Actinomicina D e Actinomicina X2 com m/z de 1277 [M + Na] e 1291 [M+Na]. A forte atividade antibacteriana apresentada pelas substâncias isoladas torna este produto uma importante fonte de compostos antibacterianos naturais

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T15:13:44Z (GMT). No. of bitstreams: 1 Costa_LuizAntonioMendoncaAlvesda_D.pdf: 24416441 bytes, checksum: bec02c6b51213d77af83bf869644c046 (MD5) Previous issue date: 2005 / Resumo: O presente trabalho foi dividido em dois projetos: a avaliação do potencial biocatalítico de microrganismos isolados da abelha Trigonna sp e o estudo de biorremediação de ambiente contaminado por Alachlor®. A atividade catalítica de oxidação de sulfeto e a hidrólise de éster sulfínico de 12 linhagens de fungos foi avaliada durante a primeira parte deste trabalho, dentre das quais, 8 linhagens foram isoladas do corpo da abelha Trigonna sp em trabalhos anteriores do nosso grupo. Os melhores microrganismos na oxidação enantiosseletiva do etil fenil sulfeto foram os Fungo CCT 5553 e Cladosporium sp. CBMAI 0210 que produziram o (S)-etil-fenil-sulfóxido (ee > 99%) e (R)-etil-fenil-sulfóxido (ee 97%), respectivamente. O (S)-etil-fenil-sulfóxido (ee > 92%) foi aplicado na síntese da S-(+)-4-metil-3-heptanona, feromônio de alarme da formiga do gênero Atta, mas uma racemização durante a eliminação do grupo sulfinila impossibilitou a síntese total. Para a resolução enzimática de (±)-benzenossulfinato de cicloexila foi selecionado os fungos Penicillium sp. CBMAI 0208 e Aspergillus ochraceus CBMAI 0211 ambos fornecendo os produtos com excessos enantioméricos > 99%. Na segunda etapa, avaliou-se a capacidade de degradação do pesticida Alachlor® de 6 linhagens de bactérias (Streptomyces sp.) e as estruturas dos produtos de degradação foram sugeridas baseados em seus padrões de fragmentação. Entre esses 8-etil-quinolina e N-metil-8-etil-indol nunca foram citados nos estudos de biodegradação do Alachlor®. / Abstract: The work presented in this thesis is divided into two projects: the evaluation of the biocatalytic potential of microorganisms isolated from Trigonna bee and those deposited in two brazilian collections and bioremediation of Alachlor contamined soil. The sulfide oxidation and sulfinic esters hydrolysis catalytic activity was screened using 12 different fungi strains, 8 of which were previously isolated from a Trigonna sp. bee. The best microorganisms for the enantioselective oxidation of ethyl phenyl sulfide were Fungus CCT 5553 and Cladosporium sp. CBMAI 0210 WHICH PRODUCED (R)-ethyl phenyl sulfoxide (ee 97%) and (S)-ethyl phenyl sulfoxide (ee > 99%) respectively. The chiral (S)-ethyl phenyl sulfoxide which was applied in the synthesis of S-(+)-4-methyl-3-heptanone, ant alarm pheromone (genus Atta), of but racemization during sulfinyl group elimination step precluded the total asymmetric synthesis. For the enzymatic resolution of the cyclohexyl (±)-benzenosulfinate we have selected Penicillium sp. CBMAI 0208 and Aspergillus ochraceus CBMAI 0211 both with the capacity of resolving the sulfinate in over 99 enantiomeric excess. In the second part, the Alachlor® degradation potential of 6 bacterium strains (Streptomyces sp.) was evaluated and the structures of biodegradation products were suggested based on their mass fragmentation patterns. Among these 8-ethyl-quinoline and N-methyl-8-ethyl-indole have never been mentioned as Alachlor biodegradation products before. / Doutorado / Quimica Organica / Doutor em Quimica

Os actinomicetos são bactérias Gram positivas caracterizadas por sua habilidade em formar hifas, são amplamente distribuídos no ambiente e conhecidos pela diversidade na produção de moléculas biologicamente ativas. O presente trabalho teve por objetivo avaliar a atividade de compostos produzidos por quarenta isolados de actinomicetos contra isolados clínicos de Enterococcus sp, Staphylococcus aureus e Staphylococcus epidermidis. O perfil de suscetibilidade das amostras clínicas foi avaliado empregando a técnica de disco difusão em ágar. A atividade antimicrobiana dos actinomicetos foi avaliada pela técnica da dupla camada. Os isolados que apresentaram atividade foram cultivados em caldo amido caseína à temperatura de 30ºC por sete dias, com agitação constante. Após o crescimento, a cultura foi filtrada para obtenção do extrato bruto. A atividade antibiótica do extrato foi avaliada através da técnica de difusão em poço. O isolado que apresentou maior espectro de ação foi selecionado para otimização dos compostos. A otimização da produção dos compostos com atividade antibiótica foi realizada através da avaliação de curva de produção, variação da fonte de carbono, tempo de incubação, pH tamponado e pH não tamponado. No ensaio de sobrecamada os isolados 50 e 8S apresentaram atividade contra a 90% das amostras de microrganismos clínicos de Staphylococcus sp. e Enterococcus sp. No ensaio de difusão em poço o isolado 50 apresentou maior atividade antibiótica que o isolado 8S. Na otimização do extrato as melhores condições de produção foram: 72 h de crescimento, fonte de carbono amido e sem tamponamento de pH. Não foi observada influência de biomassa na produção dos compostos. A cromatografia em camada delgada revelou a presença de duas bandas com fator de retenção de 0,28 (Rf1) e 0,57 (Rf2). / Actinomycetes are Gram positive bacteria, characterized by their ability to form hyphae. They are widely distributed in the environment and known for their diversity in producing biological active molecules. This study aimed to evaluate the activity of compounds produced by forty isolates of Actinomycetes against clinical isolates of Enterococcus sp, Staphylococcus aureus and Staphylococcus epidermidis. The susceptibility profile of the samples was evaluated using the disk diffusion technique in agar. The antimicrobial activity of actinomycetes was assessed by means of the double layer. Isolates that showed activity were grown in starch casein broth at a temperature of 30 ºC for seven days, with constant agitation. After growth the culture was filtered to obtain a crude extract. The antimicrobial activity of the extract was evaluated by the well diffusion technique. The actinomycete that showed activity against most of the test samples was selected for optimization(s) of the compound(s) production. The optimization of the production was performed by evaluating: growth curve, the use of different carbon source, changes in the incubation time, and culture media with buffered and unbuffered pH. In the overlay assay isolates 50 and 8S presented activity against most of Staphylococcus sp. and Enterococcus sp samples. In diffusion assay isolate 50 showed higher antibiotic activity than the isolated 8S. Compiling the results the best production conditions were: 72 h of growth, carbon source starch without pH buffering at 30°C. There was no effect of biomass (s) compound (s) activity. The thin layer chromatography revealed the presence of two bands with a retention factor of 0.28 (Rf1) and 0.57 (Rf2).

>Magister Scientiae - MSc / Lignocellulose has a complex structure composed mainly of lignin, hemicellulose and cellulose. Several enzymes are needed for the degradation of lignocellulose into simple sugars. Actinomycetes are known to produce laecases which are able to degrade lignin. Laccase activities were detected in actinomycete strains MS26 isolated from soil collected from the Zambian Copperbelt and DFNR17 isolated from soil collected from a New Zealand farm. Morphological .studies showed that the strains produced extensively branched substrate mycelia and aerial hyphae. Micromorphological characteristics were consistent with the assignment of these strains to the genus Streptomyces. Isolates were found to be mesophiles, with growth occurring in a temperature range of 16 and 45°C. Optimal growth occurred at temperatures between 30 and 37°C. Analysis of the 16S rRNA gene sequences of the strains showed that strain MS26 had the highest sequence similarity (99%) to Streptomyces atrovirens strain NRRLB-16357 and Streptomyces viridodiastaticus strain IFO 13106. Strain DFNR17 had the highest 16S rRNA gene sequence similarity (99%) to Streptomyces althioticus strain KCTC9752. The strains shared several physiological and biochemical characteristics with their closest neighbours which, along with 16S rRNA gene sequences analysis, confirmed that the strains were members of the genus Streptomyces. Attempts to identify the laecase genes from these isolates by screening a fosmid library failed. Subsequently isolates were screened by PCR using laccase-like cooper oxidase degenerate primers designed from several Streptomyces strains. A 300 bp amplicon was obtained from both isolates. Phylogenetic analysis was performed and both amplicons from strains MS26 and DFNR17 had the highest similarities with the copper oxidase gene from Streptomyces griseoflavus strain Tu4000. Therefore it is probable that the laecase activity observed for these strains is due to the activity of copper oxidase gene products.

<p>The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. <em>Streptomyces </em>the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of <em>Streptomyces coelicolor, </em>a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In <em>Streptomyces coelicolor,</em> a pleiotropic regulator belonging to the two-component system family, <em>afsQ1</em>, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered <em>afsQ1</em> allele (named <em>afsQ1*</em>), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, <em>afsQ1* </em>was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed <em>afsQ1*- </em>induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]²⁺<strong> </strong>and 782 [M+H]⁺<strong> </strong>from the strain WAC00263. 1082 [M+2H]²⁺,<strong> </strong>a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant <em>Enterococcus</em> ATCC# 51299, a clinical isolate of methicillin resistant <em>Staphylococcus aureus</em>, and a clinical isolate of <em>S. aureus</em> BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen <em>Acinetobacter baumannii</em> B0098426R and a virulent strain of the fungus <em>Cryptococcus neoformans </em>H99<em>. </em>The second newly expressed molecule, 782 [M+H]⁺<strong> </strong>was not as potent as 1082 [M+2H]²⁺,<strong> </strong>so<strong> </strong>far only exhibited antimicrobial activity against the Gram-positive laboratory strains <em>Bacillus subtilis</em> #168 and <em>Micrococcus luteus</em>. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, <em>afsQ1*</em>, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.</p> / Master of Science (MSc)

River ecosystems across the US and globally face numerous stressors that impact both ecological function and water quality. In 2015-16, municipalities along the Dan River in southern Virginia experienced repeated taste and odor (TandO) issues in their drinking water that originated from the river source water. Given that the source of TandO issues during these events were unknown, this research aimed to identify patterns in the distributions of river microorganisms that could help identify potential biological causes. Monthly water, sediment, and periphyton samples were collected for a full year from the Smith and Dan Rivers to quantify actinomycete, fungi, and chlorophyll a concentrations, which have historically been linked to TandO problems, and to characterize changes in microbial community structure. Although no significant TandO event occurred during the study period, the work produced unique and valuable data that describe patterns of microbial populations and communities in a river ecosystem. Results from the study show the abundances of actinomycetes, fungi, and chlorophyll a expressing seasonal and regional variation by habitat. From a broader ecological perspective, microbial communities sampled from water, sediment, and periphyton were each unique from each other regardless of river reach and season sampled. Overall, this research adds to our understanding of river ecology by detailing the microbial abundance and diversity in three river habitats, including periphyton, that can be used to predict sources of river TandO in future events, and offers new questions regarding how microbial diversity changes over space and time. / Master of Science / In 2015-16, cities along the Dan River in Virginia experienced multiple taste and odor (T&O) events that led to earthy and musty odors in drinking water. As part of a larger project looking at a range of possible chemical and biological sources of T&O, this research aimed to identify changes in abundance of river microorganisms that might indicate potential biological causes to T&O events. Monthly samples of water, sediment, and algal growth were collected for a year from 12 sites on the Smith and Dan Rivers. Samples were analyzed for abundances of three known T&O causing groups of organisms—actinomycetes, fungi, and photoautotrophs—and to characterize changes in total microbial communities as an indicator of ecological change occurring along the rivers. Although a significant T&O event did not occur during the study period, the research produced valuable descriptions of how important microorganisms change in a freshwater ecosystem. Actinomycetes elevated in the lower Dan River during fall, fungi elevated during the spring, and chlorophyll a was highest in the upper Smith River during winter, suggesting that photoautotrophic growth was more likely to be linked to previous T&O events. The diversity and makeup of the microbial communities in the rivers was primarily dependent on where they were growing (water, sediment, or periphyton) and secondarily on the season or the river reach. Combined, these results will help to identify causes of future T&O events in the Dan River and also provide new insights into ecological patterns of microorganisms in river ecosystems.

Actinomycetes and fungi were successfully isolated from both New Zealand and Malaysian marine invertebrates and classified as facultatively marine based on their ability to grow on both sea water and non-sea water media. Most of the extracts obtained from selected isolates were cytotoxic. A clear preference of the actinomycetes for solid-state fermentation was observed, however, for fungi no significant preference was seen. Three isolates of Streptomyces spp., four Penicillium spp. and two Paecilomyces spp. whose extracts showed good cytotoxicity were selected for further investigation. A small-scale extract obtained from a solid culture of Streptomyces sp. (LA3L2) showed good cytotoxicity and a new cytotoxic metabolite was isolated from a large-scale extract of Streptomyces sp. (LA3L2). This metabolite was characterized as S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15) and is only the third compound reported to contain the S-methyl benzothioate group. Two known compounds, montagnetol (5.16) and erythrin (5.18), were isolated from a further large-scale cultivation of Streptomyces sp. (LA3L2) and is the first reported actinomycete to produce these lichen-related compounds. In addition, two known inactive metabolites (bohemamine (5.1) and bohemamine B (5.2)) were identified from the small-scale extract. Streptomyces sp. (LA3L2) was also investigated for the effect of temperature and salinity on growth and cytotoxicity and shown to produce bohemamine only at 20 - 28℃ and 4% sea salt concentration on solid media. This isolate gave a low yield of active metabolite under all conditions. Small-scale extracts of two other Streptomyces spp. yielded three known cytotoxic metabolites. These were thiazostatin B (7.14) from Streptomyces sp. (LA5L4) and chromomycin A2 (7.1), chromomycin A3 (7.2) and chromomycin 02-3D (7.3) from Streptomyces sp. (LA3L1). All four Penicillium spp. produced known metabolites. Penicillium sp. (LY1L5) yielded two known metabolites, cycloaspeptide A (7.4) and α-cyclopiazonic acid (7.5). α-Cyclopiazonic acid (7.5) and three other known metabolites (roquefortine A (7.6), cyclopeptin (7.7) and viridicatin (7.8)) were isolated from Penicillum sp. (KK3T23). Penicillium sp. (KK3T8) produced brefeldin A (7.10), while mycophenolic acid (7.12) and brevianamide A (7.11) were produced by Penicillium sp. (KK4T14b). The effect of salinity on growth and cytotoxicity was investigated for the two Penicillium isolates producing the cytotoxic metabolite, α-cyclopiazonic acid (7.5). Saline conditions were not required for growth but metabolite production differed between the two isolates with respect to salinity. Isolate LY1L5 required saline conditions for α-cyclopiazonic production whereas isolate KK3T23 produced the metabolite under non-saline conditions and in concentrations of sea salt up to 6%. Three known compounds, indole-3-carboxylic acid (7.15), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16) were identified from Paecilomyces sp. (PR5L9). Investigation of a small-scale extract obtained from a solid culture of another Paecilomyces sp. (PR10T2) resulted in the isolation and characterization of a unique structure of a symmetrical cyclic depsipeptide, epi-angolide (NAM 6-1). NAM 6-1 was considered as a new compound based on four homoisomeric configurations (A1, A2, A3 and A4). The value of dereplication procedures with respect to the rapid identification of metabolites and enhancement of in-house metabolite libraries is discussed. Structural elucidation of nine known metabolites (7.1, 7.2, 7.3, 7.5, 7.6, 7.7, 7.8, 7.10 and 7.11) was greatly aided by the in-house dereplication techniques using LC-MS-UV and AntiMarin database. A significant advantage was gained by the use of the CapNMR which enabled NMR characterization of very small quantities of metabolites (<20 µg). Approximately <5 µg of materials were required to perform 1D proton NMR experiments for the dereplication of seven known compounds; bohemamine (5.1), bohemamine B (5.2), thiazostatin B (7.14), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16). Approximately 20 µg of materials were needed to acquire 1D and 2D (HSQC, HMBC and NOE) NMR spectra for structural elucidation of the new metabolite, S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15). Some 8 µg of materials were sufficient to perform 1D and 2D (COSY, HSQC and HMBC) NMR experiments for complete structural characterization of two known metabolites, montagnetol (5.16) and erythrin (5.18). Approximately 10 µg of materials were needed to acquire 1D and 2D NMR (COSY, HSQC and HMBC) experiments for structural elucidation of the new compound, epi-angolide NAM 6-1 (A1, A2, A3 and A4). Rapid identification of known fungal metabolites enabled the in-house HPLC-UV/Rt library to be enhanced by eight metabolites (7.5, 7.6, 7.7, 7.8, 7.10, 7.11, 7.17 and 7.16). An HPLC-UV/Rt library for actinomycete metabolites was successfully established with the insertion of eight known metabolites (5.1, 5.2, 5.16, 5.18, 7.1, 7.2, 7.3 and 7.14).

Vingt souches d'actinomycètes ont été isolées à partir d'échantillons de sol et de bois prélevés à partir d'une vielle maison dans l'ancienne médina de Fès et d'échantillons de sol prélevés de la région de Moulay Yacoub et des rives d'Ouèd Sebbou. L'activité antimicrobienne, réalisée par la technique des cylindres d'agar et celle des stries croisées, a été déterminée contre des bactéries à Gram positif (Bacillus subtilis 5262, Bacillus cereus cip 14579, Staphylococcus aureus 7625, Staphylococcus epidermidis 6821) ; des bactéries à Gram négatif (Pseudomanas aeroginosa 76110, E.coli cip 7624) et la levure Candida albicans. Parmi les vingt isolats, douze (60 %) ont montré une activité contre au moins une des souches tests. Par ailleurs, l'analyse moléculaire des vingt isolats par amplification et séquençage partiel du gène de l'ARNr 16S a permis d'attribuer dix huit isolats au genre Streptomyces et deux aux genres Saccharothrix et Lentzea. De plus, le séquençage de la quasi-totalité de l'ADNr 16S (1460 pb) de cinq isolats (Sj32, Sj33, Sj38, Sj68 et Sj69) nous a permis d'assigner les isolats Sj38 et Sj69 à S. parvus, et Saccharothrix sp., respectivement. Les substances bioactives produites par les cinq souches d'actinomycètes sont extraites par des solvants organiques et les molécules produites sont purifiées par HPLC. Une étude de la stabilité de l'activité antibactérienne en fonction de la température et de la protéinase K a montré que les substances bioactives seraient de nature non protéique pour Sj32, Sj33, Sj68 et Sj69 et protéique pour Sj38. Une étude de la cytotoxicité de la fraction active de la souche Sj32 sur les cellules MRC-5 a montré qu'elle présente une forte cytotoxicité contre ces cellules. Enfin, l'étude de l'effet de la fraction active de la souche Sj32 sur la transcription et la traduction bactérienne in vitro, a montré que cette substance purifiée n'inhibe pas ces deux processus biologiques. / Twenty strains of actinomycetes were isolated from soil and wood samples taken from an old house in the old medina of Fez and also soil samples from the region of Moulay Yacoub and banks of Sebbou River. The antimicrobial activity, carried out using the agar piece and cross striations methods, were tested against Gram-positive bacteria (Bacillus subtilis 5262, Bacillus cereus cip 14579, Staphylococcus aureus 7625, Staphylococcus epidermidis 6821); bacteria Gram-negative (Pseudomonas aeruginosa 76110, E. coli cIP 7624) and the yeast Candida albicans. Among the 20 isolates, 12 (60%) showed activity against at least one of the test strains.Molecular analysis of the twenty isolates by amplification and partial sequencing of the 16S rRNA gene allowed us to assign eighteen isolates to the genus Streptomyces and two to genera Saccharothrix and Lentzea.In addition, sequencing of nearly complete 16S rDNA (1460 bp) of five isolates (Sj32, Sj33, Sj38, Sj68 and Sj69) allowed us to assign isolates Sj38 and Sj69 to S. parvus and Saccharothrix sp., respectively.Bioactive substances produced by the five strains of actinomycetes were extracted by organic solvents and the molecules produced are purified by HPLC. A study of the stability of the antibacterial activity according to temperature and proteinase K showed that the bioactive substances are of non-protein nature for Sj32, Sj33, Sj68 and Sj69 and of protein nature for Sj38.A study of the cytotoxicity of the active fraction of the strain Sj32 on MRC-5 cells showed that it had a strong cytotoxcity against these cells.Finally, the in vitro study of the effect of the active fraction of the strain Sj32 on bacterial transcription and translation, showed that the purified substance didn't inhibit these two biological processes

A utilização de técnicas moleculares têm facilitado o estudo de comunidades bacterianas complexas no ambiente. O presente trabalho teve como objetivo utilizar a técnica DGGE para avaliação dos efeitos do aumento da temperatura, causado pela queima de um canavial, na estrutura da comunidade bacteriana de solo, com ênfase ao grupo dos actinomicetos. Foram coletadas amostras de solo em diferentes profundidades, antes e depois da queima, e dados físico-químicos e climáticos associados. O DNA da comunidade bacteriana foi amplificado utilizando conjunto de primers específicos para o Domínio Bacteria e para o grupo de actinomicetos, e os produtos de amplificação analisados por DGGE. Resultados obtidos para as populações de actinomicetos não foram conclusivos. Apesar da variação dos parâmetros físico-químicos do solo provocadas pela queima, os padrões de bandas obtidos com os primers para o Domínio Bacteria, apresentaram-se uniformes. Sendo assim, nas condições de estudo deste trabalho, os resultados obtidos não revelaram alterações na estrutura da comunidade bacteriana do solo de canavial depois da queima. / The utilization of molecular techniques has facilitated the study of complex bacterial communities in the environment. The present study aimed at using DGGE technique to evaluate the effect of temperature variation, caused by sugar-cane plantation burn, in the soil bacterial community structure emphasizing the actinomycete group. Soil samples from different depths, were collected before and after the bum, as well as physical-chemical and climatic associated data. The bacterial community DNA was amplified using a specific primer set and the amplification products analyzed by DGGE. The results obtained for the actinomycete populations were not conclusive. Despite the variation of the soil parameters caused by the burn, the band patterns obtained used in this study were uniform. Therefore, under the conditions used in this study, the results obtained did not show any alteration in the structure of soil bacterial community associated with sugar-cane plantation after the burn.