Göttingen, Universiẗat, Diss., 2000.

Thesis (Ph.D.)--University of Idaho, May 2006. / Major professor: Donald L. Crawford. Abstract. Includes bibliographical references. Also available online in PDF format.

Universiẗat, Diss., 2004--Freiburg (Breisgau).

University, Diss., 2005--Jena.

Whole-genome sequencing has shown that the large (8-12 Mbp) genomes of Streptomyces and allied genera of Gram-positive filamentous bacteria house a rich and previously underestimated repertoire of gene clusters for biosynthesis of specialised metabolites, including antibiotics, immunosuppressants and anticancer compounds. Many of these clusters remain uncharacterised because they are not expressed under the culture conditions used. Even for strains from which a specific compound has been identified, the challenge remains to link the compound to its gene cluster, and to develop procedures for analysing and manipulating the biosynthetic pathway. In this work, three strains have been studied that address different aspects of this challenge. Streptomyces sp. DSM4137 is a genetically amenable strain and a notably prolific producer of diverse natural products, but also has multiple biosynthetic gene clusters that remain uncharacterised. In an attempt to differentiate those clusters where the genes are expressed from those that are essentially silent, the transcriptome of DSM4137 was analysed using total RNASeq and the results were used to inform analysis of HPLC/MS data of extracts under the same conditions. There was shown to be good correlation between the RNASeq results and the pattern of metabolites produced, suggesting that RNASeq may be a useful complement in the search for novel gene clusters. In contrast, Saccharopolyspora spinosa, producer of the valuable insecticidal spinosyns, is not genetically amenable. A new technique has been developed for the mobilisation of an entire biosynthetic gene cluster and refactoring attempted to increase the production of spinosyns in a heterologous strain. Total transcriptome was analysed by RNAseq to give an insight into the regulation of the WT strain, helping identify future methods for strain manipulation for increase yields.

Streptomyces are interesting gram positive filamentous bacteria and have been studied mostly in the context of antibiotic production. This system is controlled by specific networks of genes through regulatory signals that combine to regulate both morphological and physiological differentiation in the organism. But it is in the context of growth and cell division that they are also fascinating. FtsW is one of four Shape, Elongation, Division and Sporulation (SEDS) family proteins encoded in the genome of S. coelicolor. In this study the effect of ftsW complementation, and its overexpression in S. coelicolor, and role of other related proteins was studied. Complementation of an ftsW mutant resulted in a sporulating phenotype confirming that the white phenotype was due to ftsW disruption, and ftsW function was restored by the complementing plasmid. Macroscopically, fts W or ^/com plem entation or overexpression in S. coelicolor does not show any difference in phenotype. In addition, the microscopic analysis revealed that there is no effect of ftsW or ftsl overexpression on the sporulation septation and chromosomal condensation of aerial hyphae. FtsW localization was investigated revealing a diffuse distribution of fluorescence in the aerial hyphae of an ftsWcomplemented strain, due to in vivo proteolytic cleavage of the FtsW-mCherry translational fusion protein, precluding any inference of FtsW localisation itself. Also mycobacterial orthologs of FtsW and Ftsl cannot replace the function of their related streptomycete proteins. By construction of triple mutants of ftsW, sfr and rodA2 SEDS genes in one strain of S.coelicolor, results showed that these genes are dispensable for growth and survival. No difference was observed between the triple mutant strain that exhibited a white non-sporulating phenotype, and the fts W single mutant phenotype. An absence of sporulation septa in both the triple mutant and ftsW single mutant aerial hyphae, while an sfrlrodA2diO\xb\Q mutant has normal sporulation septa, confirming that FtsW is required specifically for sporulation septation. In contrast and from the results of fluorescence microscopical analysis of the vegetative mycelium of the triple mutant strain and the wild type strain, a similar staining pattern of vegetative septa were observed, suggesting that these genes (ftsW, sfr and rodA2) are not required for vegetative septation in S. coelicolor. In addition, in order to understand the cell division mechanism in S. coelicolor more clearly, the Bacterial Adenylate Cyclase Two- Hybrid (BACTH) system used to study protein-protein interactions. Proteins tested were FtsW, Ftsl, FtsZ, FtsQ, CrgA, Sfr, MreB, RodA, RodA2, penicillin binding proteins PBP1, PBP2 and PBP3) in several combinations of protein pairs. Notably, the results showed a strong interaction between CrgA and FtsQ, and also with other cell division proteins, suggesting aivcentral role for CrgA in the cell division process. According to this significant results, alignment of the S', coelicolor CrgA sequence with orthologs from other Actinobacteria was carried out, revealing only four amino acids G4 0, W45, N65 and Ws3 that are well conserved. After site directed mutagenesis to modify S. coelicolor CrgA, this revealed that the amino acids N65 and Wg3 are required for interaction of the protein with itself and PBP2.

Made available in DSpace on 2014-06-11T19:27:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2005-12-15Bitstream added on 2014-06-13T19:14:52Z : No. of bitstreams: 1 favarin_ec_me_rcla.pdf: 436560 bytes, checksum: 985e56580dc75e41c059cd89ca7f4655 (MD5) / A simbiose entre as formigas cortadeiras e seu fungo originou-se a aproximadamente 50 milhões de anos. A diversidade biótica dos formigueiros, no entanto, não se restringe apenas à formiga e ao fungo simbionte. Recentemente foi descoberto um terceiro mutualista, uma bactéria filamentosa do grupo dos actinomicetos, cuja principal função seria a inibição do crescimento de parasitas do jardim de fungos, especialmente o fungo conhecido como Escovopsis sp, através da produção de antibióticos. O presente trabalho teve como objetivos verificar o potencial das linhagens de Streptomyces, que foram isoladas de Attini, como produtoras de substâncias antimicrobianas frente aos fungos Escovopsis weberi e outros microrganismos e também a caracterização dessas linhagens através de técnicas de biologia molecular. Foram selecionadas nove linhagens de Streptomyces, que foram cultivadas em meio SCN líquido, para a obtenção dos filtrados. Para os ensaios de antibiose envolvendo os fungos Escovopsis weberi, foram testadas diferentes concentrações de filtrados adicionadas ao meio A sólido e foi verificada a porcentagem de germinação dos conídios em cada concentração. Para a determinar a atividade dos filtrados frente as bactérias e leveduras, os testes foram realizados pelo método de difusão em agar. Os resultados mostraram que, mesmo variando os filtrados obtidos das linhagens de Streptomyces, a inibição da germinação dos conídios foram muito semelhantes, e todas as linhagens do fungo Escovopsis weberi apresentaram uma inibição homogênea frente a mesma concentração de filtrado. Com relação as bactérias e leveduras, os resultados mostraram que houve diferenças na intensidade da resposta. Algumas linhagens inibiram o crescimento de todas as bactérias e leveduras testadas, outras não inibiram apenas algumas culturas e teve linhagens que não inibiram nenhuma das... / The symbiotic relationship between leaf-cutting ants (Tribe Attini) and their mutualistic fungus probably arose fifty million years ago. However, these two organisms are at least, part of the biological diversity found in nests of these insects. Recently, it was discovered a third mutualist, an antibioticproducing actinomycete, which is used by the ants to control the development of garden parasites, specially within the microfungus genus Escovopsis sp. In order to determine which isolates of this actinomycete could affect the growth of Escovopsis weberi and other microorganisms; nine strains of Streptomyces sp. were grown in liquid SCN and the resultant media (extract) were filtered and used in the experiments. Also, all actinomycete strains were characterized by molecular sequencing of the 16 rDNA region. In the assays involving E. weberi, different extract amounts were added into solid medium (Meio A) and the conidial germination rate were determined after incubation. Disk-difusion method were used to verify the antimicrobial activity of these extracts over a large range of bacteria and yeasts. In spite of the concentration used in E. weberi assays, inhibiton of spore germination was achieved and this response was similiar among E. weberi isolates. On the other hand, bacteria and yeasts demostrated a high degree variability in this response. Some Streptomyces sp. strains inhibited all bacteria and yeasts tested, but other just inhibited a few of them. The molecular sequencing of the 16S rDNA region have shown that all actinomycete strains used in this work were grouped with other Streptomyces species found in GenBank. In spite of phylogenetic analyses have grouped Attini isolates in different clades, the activity of the antimicrobial compounds produced by these bacteria had a high degree of homogenicity over E. weberi... (Complete abstract, click electronic address below)

Made available in DSpace on 2014-06-12T15:50:36Z (GMT). No. of bitstreams: 2 arquivo4461_1.pdf: 581325 bytes, checksum: 3ac6df73d6c58a450eea979f2d9f9829 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Com a utilização clínica de agentes antimicrobianos começaram a surgir bactérias resistentes a diversos antibióticos, entre os quais, se destacam os β- lactâmicos. A hidrólise do anel β-lactâmico pelas enzimas β-lactamases é o mecanismo de resistência bacteriana mais bem documentado. A obtenção de Streptomyces produtores de inibidores de tais enzimas constitui uma importante estratégia para contornar o problema da resistência bacteriana, garantindo a continuidade da terapia antimicrobiana com os β-lactâmicos. Com este objetivo, 19 linhagens de Streptomyces spp, pertencentes a Coleção de Microrganismos do Departamento de Antibióticos da UFPE (DAUFPE), isoladas de solos e preservadas em óleo mineral foram testadas quanto a capacidade de inibir a ação de β-lactamases produzidas por Klebsiella aerogenes ATCC 15380. As 19 linhagens foram purificadas utilizando-se os meios ISP-1 e TSB e submetidas à seleção primária pelos métodos de difusão em ágar, utilizando bloco de gelose e cultivo em meio sólido nos meios CAA e ISP-2. No meio CAA, 15% das linhagens formaram halo de inibição, com 10 a 12 mm de diâmetro e 10% formaram halos maiores que 20 mm de diâmetro. No meio ISP- 2, 5% das linhagens formaram halo de 12 mm de diâmetro e 11% halos de 17 mm de diâmetro. A partir destes ensaios, cinco linhagens (DAUFPE 3036, DAUFPE 3060, DAUFPE 3094 DAUFPE 3098 e DAUFPE 3133) que formaram os maiores halos de inibição, foram selecionadas para ensaio em meio líquido usando meios de cultura sintéticos ou quimicamente definidos e complexos. As fermentações foram realizadas a 300C e 200rpm durante 96 horas. As amostras retiradas em intervalo de tempo pré-definido foram submetidas à centrifugação a 11000 rpm por 5 minutos, para a separação do líquido metabólico, o qual foi submetido a determinação de pH, atividade antimicrobiana e ácido clavulânico, sendo este um potente inibidor de β- lactamases. Observou-se que o melhor meio para produção de inibidor de β- lactamase foi o MPE modificado, em que todas as linhagens apresentaram atividade antimicrobiana contra Klebsiella aerogenes ATCC 15380. Com relação à produção de ácido clavulânico, três linhagens (DAUFPE 3036, DAUFPE 3060 e DAUFPE 3094) apresentaram resultado positivo em dois meios de cultura (MPE modificado e meio com glicerol), enquanto que as duas linhagens restantes (DAUFPE 3098 e DAUFPE 3133) produziram outros inibidores

Submitted by Pedro Barros (pedro.silvabarros@ufpe.br) on 2018-07-31T22:40:20Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Elizianne Pereira Costa.pdf: 1981689 bytes, checksum: 0b2afb4e38bafdf78edf5fbca37ec218 (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-08-02T17:26:09Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Elizianne Pereira Costa.pdf: 1981689 bytes, checksum: 0b2afb4e38bafdf78edf5fbca37ec218 (MD5) / Made available in DSpace on 2018-08-02T17:26:09Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) TESE Elizianne Pereira Costa.pdf: 1981689 bytes, checksum: 0b2afb4e38bafdf78edf5fbca37ec218 (MD5) Previous issue date: 2017-02-23 / FACEPE / Proteases microbianas são muito utilizadas na indústria, tendo papel chave no desenvolvimento de diversos processos biotecnológicos. As colagenases são um grupo de proteases que degradam a tripla hélice do colágeno e são utilizadas na indústria farmacêutica, do couro e no amaciamento de carnes. O objetivo do presente trabalho foi identificar a linhagem Streptomyces sp. através de características morfológicas, bioquímicas e técnicas moleculares, avaliar a produção de proteases em diversos substratos (gelatina, resíduo de café, farinha de soja, farelo de trigo e penas de galinha), produzir e purificar colagenase obtida a partir de fermentação submersa, caracterizar a colagenase quanto à influência da temperatura, pH, íons metálicos, surfactantes e inibidores enzimáticos, produzir peptídeos a partir da degradação de colágeno tipo I e tipo V, verificar o potencial anticoagulante dos peptídeos obtidos, e avaliar a cinética e termodinâmica da hidrólise de azocoll pela colagenase purificada. A actinobactéria foi identificada como Streptomyces antibioticus UFPEDA3421, e apresentou produção de enzimas proteolíticas quando cultivada em fermentação submersa em todos os substratos testados. As maiores atividades enzimáticas obtidas foram: protease (46,62 U/mL) em meio contendo farinha de soja, queratinase (32,96 U/mL) em meio contendo penas de galinha e protease fibrinolítica (9,3 U/mL) em meio com farinha de soja. Essas proteases não apresentaram afinidade pela resina de troca iônica e foram coletadas na fração não adsorvida. A colagenase foi produzida nos cinco substratos, apresentando maior atividade em meio contendo resíduo de café (377,50 U/mL), a partir do qual foi purificada por cromatografias de troca iônica e exclusão molecular, com atividade colagenolítica de 150,50 U/mL, atividade específica de 16.723,00 U/mg, fator de purificação de 136,7 e massa molar de 41,28 kDa. A caracterização da colagenase demonstrou que a enzima é uma metaloprotease neutra, com máxima atividade a 60 °C, pH 7.0, e possui afinidade por íons metálicos mono- e di- valentes, além de ser potencializada na presença dos surfactantes Tween 20, Tween 80 e Triton X-100. A degradação de colágeno tipo V produziu peptídeos com atividade anticoagulante que retardaram o tempo de coagulação sanguínea em 88s em comparação ao controle. Na hidrólise do azocoll, os parâmetros cinéticos obtidos da colagenase foram: Km = 27,14 mg/mol; Vmax = 714,29 mg/mol/min; Kcat = 79,9 s⁻¹, e; Kcat / Km = 2,95 ml/mg.s. Os parâmetros termodinâmicos apresentaram os seguintes valores (a 25 °C): Energia de Ativação (Ea) = 65,224 KJ/mol; Entalpia (ΔH) = 62,75 KJ/mol; Entropia (ΔS) = 1,96 J/mol; Energia Livre de Gibbs (ΔG) = 62,16 KJ/mol; Energia Livre na Ligação ao Substrato (ΔGᴇ₋s) = 8,18 KJ/mol, e; Energia Livre no Estado de Transição (ΔGᴇ₋ᴛ) = -2,64 KJ/mol. Os dados mostram potencial na produção de proteases, especificamente de colagenase, em diversos substratos pela linhagem Streptomyces antibioticus UFPEDA3421, em especial no cultivo em meio de cultura contendo resíduo de café, um resíduo abundante no Brasil. A colagenase purificada apresenta características que a qualificam para uso na produção de peptídeos bioativos. / Microbial proteases are widely used in industry, playing a key role in the development of various biotechnological processes. Collagenases are a group of proteases that degrade the triple helix structure of collagen and are used in pharmaceutical, leather and meat industry. The aim of the present work was to identify the strain Streptomyces sp. using morphological, biochemical and molecular techniques, to evaluate the production of proteases in various substrates (gelatin, coffee powder residue, soybean meal, wheat bran, and chicken feathers), to produce and purify collagenase obtained from submerged fermentation, to characterize the influence of temperature, pH, metallic ions, surfactants and enzymatic inhibitors on collagenase activity, produce peptides from degradation of type I and type V collagen, to verify the anticoagulant potential of the peptides obtained, and to evaluate the kinetics and thermodynamics of the azocoll hydrolysis by the purified collagenase. The actinobacteria was identified as Streptomyces antibioticus UFPEDA3421, and showed proteolytic enzyme production when grown in submerged fermentation in all substrates tested. The highest enzymatic activities were: protease (46.62 U/mL) in medium containing soybean meal, keratinase (32.96 U/mL) in medium containing chicken feathers and fibrinolytic protease (9.3 U/mL) in medium with soybean meal. These proteases had no affinity for the ion exchange resin and were collected in the non-adsorbed fraction. The collagenase was produced in the five substrates analyzed, showing higher activity in medium containing coffee powder residue (377.50 U/mL), from which it was purified by ion exchange and molecular exclusion chromatography, with a collagenolytic activity 150.50 U/mL, specific activity 16,723.00 U/mg, purification factor 136.7 and molar mass 41.28 kDa. The characterization of collagenase demonstrated that the enzyme is a neutral metalloprotease, with maximum activity at 60 °C, pH 7.0, has affinity for mono- and di-valent metal ions, and is potentiated in the presence of Tween 20, Tween 80 and Triton X-100 surfactants. Degradation of type V collagen produced peptides with anticoagulant activity that delayed blood coagulation time in 88s when compared to control. In the azocoll hydrolysis, the kinetic parameters obtained from collagenase were: Km = 27,14 mg/mol; Vmax = 714,29 mg/mol/min; Kcat = 79,9 s⁻¹, e; Kcat/ Km = 2,95 ml/mg.s. The thermodynamic parameters showed the following values (at 25 °C): Activation energy (Ea) = 65,224 KJ/mol; Enthalpy (ΔH) = 62,75 KJ/mol; Entropy (ΔS) = 1,96 J/mol; Gibbs free energy of activation (ΔG) = 62,16 KJ/mol; Free energy of substrate binding (ΔGᴇ₋s) = 8,18 KJ/mol, and; Free energy for transition state formation (ΔGᴇ₋ᴛ) = -2,64 KJ/mol. The data show potential in the production of proteases, specifically collagenase, in several substrates by the strain Streptomyces antibioticus UFPEDA3421, especially in culture medium containing coffee residue, a residue abundant in Brazil. Purified collagenase has characteristics that qualify it for use in the production of bioactive peptides.

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