Techn. Universiẗat, Diss., 1999--Berlin.

Techn. Universiẗat, Diss., 2001--Berlin.

Universiẗat, Diss., 2004--Frankfurt (Main).

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Streptomyces spp. are responsible for a large proportion of the world-wide quality deterioration of potatoes causing a potato tuber disease called cornmon scab. Determining the genetic diversity of the Streptomyces spp., especially the main pathogen, S. scabies, has been a prerequisite for the ultimate control of common scab. Techniques responsible for the classification and determination of genetic diversity have improved with advances in DNA technology. Analysis of South African (S.A.) S. scabies isolates has been focusing on the organisms' morphology, physiology, pathogenicity and melanin production, but the classification of S. scabies using DNA techniques has not yet been explored. In this study various DNA techniques were screened for optimal use in determining the genetic diversity within and among isolates of S. scabies. Bacteria had been sampled from the main potato producing regions in S.A. and a few other regions. The techniques explored included RAPDs, AFLPs, RAMS, Rep-PCR, 16S rDNA sequencing and ITS analysis. The first three techniques had to be abandoned due to non-reproducibility between the same isolate extracted on separate occasions and ITS analysis was abandoned due to sequencing difficulties. Of the three Rep-PCR techniques tested (BOX, ERIC and REP), BOX was selected because it produced the clearest and most reproducible results. BOX-PCR and 16S rDNA sequencing were therefore ultimately selected as the methods to analyse the genetic diversity of the S. scabies isolates. Information concerning the pathogenicity of the isolates was supplied by the Vegetable and Ornamental Plant Research Institute of the Agricultural Research Council (VOPI, ARC, Roodeplaat). A brief analysis of the pathogenicity prediction of the isolates in this study was explored with the PCR technique. Presence of the necJ gene was previously shown to be an indication of the pathogenicity within the Streptomyces spp. group. PCR analysis is based on the amplification of a O.72kb fragment (necl) in pathogenic isolates which was absent in non-pathogenic isolates. However, in this study the test for pathogenicity lacked specificity and sensitivity and some of the problems experienced included non-reproducibility between PCR reactions and the presence of the pathogenic fragment in the nonpathogenic isolates (as designated by VOPI, ARC). These observations led to the conclusion that this technique is not an ultimate test for pathogenicity of S. scabies isolates in a South African context. The genetic distances and similarity matrices of the Rep-PCR results were calculated using Nei's genetic distance calculation (Nei M, 1975). Clusters from these matrices were constructed using the unweighted pair group average (UPGMA) with the PAUP4 package. The clusters for the 16S rDNA sequences were formed with the Neighbor Joining (NJ) method and the PAUP4 package. The NJ trees do not take small sequencing differences into account, therefore a Parsimony Network had to be constructed. The trees obtained with the 16S rDNA sequencing techniques grouped most S. scabies isolates into one major group with a 100% bootstrap robustness of this group. More genetic diversity was illustrated by the BOX-PCR technique and the isolates were generally grouped according to their different regions of origin. However, the bootstrap values were low, indicating a lack of robustness regarding the BOXPCR clustering. This was not unexpected as the number of data points employed in the BOX technique is very limited. Both techniques revealed unexpected grouping of a few isolates. Their isolated positions could be attributed to possible misclassification or to the fact that they could be genetically different S. scabies isolates. Streptomyces spp. (other than S. scabies) displayed enough differences to place them in their own distinct groups using both techniques. Comparison of the cluster results obtained in this study did not correlate to the data supplied by the VOPI, ARC (morphology, physiology, pathogenicity and melanin production) which revealed differences between the S. scabies isolates within their respective regions. The lack of diversity displayed by the 16S rDNA technique can be attributed to the fact that only a limited section of the genome is involved making it inappropriate for intra-species genetic diversity analysis. The BOX technique takes various loci within the genome but is still not ideal for a thorough genetic diversity analysis. This study represents the first attempt to determine the genetic diversity of S. scabies in S.A. on DNA level. / AFRIKAANSE OPSOMMING: Streptomyces spp. is verantwoordelik vir 'n _groot deel van die wereld afname in aartappel kwaliteit as gevolg van die aartappelknol siekte bruinskurf. Die bepaling van die genetiese diversiteit tussen die Streptomyces spp., varal die hoof patogeen in die groep, S. scabies, is 'n vooreiste vir die uiteindelike beheer van bruinskurf. Tegnieke verantwoordelik vir die klassifikasie en bepaling van genetiese diversiteit het verbeter met vooruitgang in DNA tegnologie. Analise van Suid Afrika (S.A.) se S. scabies isolate konsentreer op die organisme se morfologie, fisiologie, patogenisiteit en malanien produksie, maar die klassifikasie van S. scabies met die behulp van DNA tegnieke is nog nie uitgevoer me. In hierdie studie is verskeie DNA tegnieke ondersoek vir optimale bepaling van genetiese diversiteit binne en tussen S. scabies isolate van S.A Bakteriee is verkry van die hoof aartappel-produserende areas in S.A. en ook van 'n paar ander areas. Die tegnieke wat in die studie gebruik is, het RAPDs, AFLPs, Rep-PKR, 16S rDNA volgordebepaling en ITS analise ingesluit. Die eersgenoemde drie tegnieke is uitgesluit as gevolg van nie-herhalende resultate tussen dieselfde isolaat geisoleer op verskillende geleenthede. ITS analise is uitgesluit as gevolg van probleme met volgordebepaling. Rep- PKR en 16S rDNA volgordebepaling is uiteindelik gekies as die mees geskikte metodes vir die analise van genetiese diversiteit tussen S. scabies isolate in hierdie studie omdat albei skynbare herhaalbare resultate gelewer het. Inligting met betrekking tot die patogenisiteit van die isolate is voorsien deur die Groente en Sierplant Instituut van die Landbou Navorsinsraad (YOPI, LNR). 'n Vinnige analise van die patogenisiteits voorspelling van die isolate is uitgevoer met die PKR tegniek. Dit is voorheen aangetoon dat die teenwoordiheid van die necJ geen dui op die patogenisiteit in die Streptomyces sp _groep. PKR analise het 'n O.72kb fragment (necl) in patogeniese isolate geamplifiseer wat nie teenwoordig was in niepatogeniese isolate nie. Hierdie toets vir patogenisiteit soos gebruik in hierdie studie was egter onspesifiek en onsensitief en sommige van die probleme wat ondervind is sluit in nie-herhaalbaarheid tussen PKR reaksies en die teenwoordigheid van die patogeniese fragment in die nie-patogeniese isolaat (soos beskryf deur YOPI, LNR). Uit hierdie waarnemings word afgelei dat die tegniek nie 'n geskikte toets vir patogenisiteit van S. scabies isolate in 'n S.A konteks is nie. Die genetiese afstande en ooreenkomstige matrikse van die Rep-PCR resultate is bereken met die genetiese afstand bepaling van Nei (Nei M, 1975). Groepe is gevorm met die "unweighted pair group average" (UPGMA) en PAUP4 pakket. Die "Neighbor Joining" (NJ) groepe is gevorm met die 16S rDNA volgordebepaling data mbv die PAUP4 pakket. Die NJ groepering neem nie klein volgorde verskille in ag nie en gevolglik moes'n "Parsimony Network" opgestel word. Die groepering met die 16S rDNA volgordebepaling het meeste van die isolate in een hoof groep geplaas met 'n 100% "bootstrap" waarde. Meer genetiese diversiteit is met die BOX-PCR tegniek gevind en isolate was oor die algemeen gegroepeer vol gens van hul oorsprong. Die "bootstrap" waardes vir die BOX tegniek was baie laag. Dit was nie onverwags nie, want die hoeveelheid data punte was beperk met die BOX tegniek. Albei tegnieke het 'n aantal afwykende isolate vertoon. Hul ge-isoleerde posisies kan toegeskryf word aan moontlike misklassifikasies van die isolaat. Die moontlikheid dat daar wel genetiese verkille tussen die isolate is, kan egter nie uitgesluit word nie. Streptomyces spp. (uitgesluit S. scabies) het genoeg variasie vertoon om hulle in hul eie groepe met die gebruik van beide tegnieke te plaas. Vergelyking tussen die groepe in die studie stem nie ooreen met die data verkry vanaf YOPl, LNR (morfologie, fisiologie, patogenesiteit en melanien produksie) nie wat verskille tussen S. scabies isolate binne 'n sekere gebied vertoon. Die gebrek aan diversiteit soos vertoon deur die 16S rDNA tegniek kan toegeskryf word aan die feit dat slegs 'n beperkte gedeelte van die genoom ondersoek word, wat dit ongeskik vir intra-species genetiese diversiteit analise maak. Die BOX tegniek neem verskeie loci in die genoom in ag, maar is steeds nie ideaal vir deeglike genetiese diversiteit analise nie. Hierdie studie verteenwoordig die eerste poging om die genetiese diversiteit van S. scabies in S.A. op DNA vlak te bepaal.

Para determinar as variáveis morfogênicas, estruturais e o fluxo de tecidos os tratamentos foram duas intensidades (baixa e moderada) e dois métodos de pastejo (pastejo com lotação contínua e rotacionada). No experimento 1 o bastão graduado apresentou a melhor correlação com a massa de forragem (r2=0,65). No 2 as melhores correlações foram obtidas quando avaliadas as faixas de pós-pastejo para o disco medidor (r2=0,47) e as de pré-pastejo para o bastão graduado (r2=0,36). Para as variáveis morfogênicas e estruturais as intensidades de pastejo foram responsáveis por diferenças na taxa de elongação de folhas (intensidade baixa resultou em maior taxa de elongação) e nas características estruturais (intensidade baixa resultou em menor densidade de perfilhos, maior comprimento e número de folhas vivas). Os métodos de pastejo influenciaram as características morfogênicas (lotação contínua resultou em maior taxa de elongação de folhas, maior taxa de surgimento e tempo de vida das folhas no ciclo de observação I) e estruturais (lotação contínua resultou em maior densidade de perfilhos); bem como foi obtida interação com as intensidades e com os ciclos de avaliação. O fluxo de crescimento (favorecido por lotação rotacionada a baixa intensidade) e de senescência (favorecido por lotação contínua a baixa intensidade) foram afetados pelos tratamentos, enquanto que o fluxo de consumo não foi alterado pelos tratamentos.

O filo Actinobacteria é um importante grupo de bactérias Gram positivas amplamente distribuídas nos ambientes aquáticos e terrestres, são grandes produtores de compostos biologicamente ativos e, portanto, de grande interesse biotecnológico. O gênero Streptomyces destaca-se como maior produtor destes compostos, sendo responsável por cerca de 70% dos antibióticos que hoje utilizamos. A identificação dos organismos deste gênero ainda é um desafio. Durante muitos anos a identificação foi realizada somente com base em características morfológicas e fisiológicas. Atualmente, com o avanço das técnicas moleculares, há um grande número de espécies relatadas em bancos de dados genômicos. Este trabalho tem por objetivo identificar isolados de actinobactérias presentes no Laboratório de Microbiologia Ambiental ICBS/UFRGS com auxílio das técnicas de BOX-PCR, amplamente utilizado em estudos de diversidade dentro deste filo, e URP-PCR. E, além disso, realizar purificação parcial de um composto antimicrobiano efetivo contra bactérias produzido pelo isolado Streptomyces 8S. Os primers URP e BOX1AR produziram distintos padrões de amplificação nos isolados estudados, porém não foi possível investigar as relações de similaridade entre eles. Ainda o sequenciamento da região 16S rDNA não foi eficiente para identificar as espécies. Para a purificação do composto antimicrobiano foi realizada extração líquido-líquido com o solvente acetato de etila, posteriormente cromatografia de gel-filtração (Sephadex G-75) e troca iônica (SP-Sepharose e DEAE-celulose). A atividade antimicrobiana do composto foi recuperada após cada etapa. O composto manteve-se ativo após os ensaios de estabilidade frente à adição de EDTA, enzimas proteolíticas e altas temperaturas. Isto sugere que o composto antimicrobiano não é de origem protéica. Este trabalho sugere novos estudos a partir dos resultados preliminares obtidos, como a amplificação de todo o fragmento 16S rDNA dos isolados de actinobactérias e a investigação do composto antimicrobiano através de cromatografias de alta resolução. / The Actinobacteria phylum is an important group of Gram positive bacteria widely distributed in terrestrial and aquatic environments. They are major producers of biologically active compounds and therefore of great biotechnological interest. The genus Streptomyces stands out as the largest producer of these compounds, accounting for about 70% of the antibiotics that we use today. The identification of these organisms is still a challenge. For many years the identification was carried out only based on morphological and physiology characteristics. Today, with the advance of molecular techniques, there are a large number of species reported in genomics database. This work aims to identify isolates of actinobacterias present in the Laboratório de Microbiologia Ambiental ICBS / UFRGS with the help of BOX-PCR techniques, widely used in diversity studies within this phylum, and URP-PCR. And besides that, performing partial purification of an antimicrobial compound effective against bacteria produced by Streptomyces 8S isolated. The URP and BOX1AR primers produced different amplification patterns in the isolates, but it was not possible to investigate the relationship of similarity between them. Also the sequencing of 16S rDNA was not efficient to identify the species. To purify the antimicrobial compound was carried out liquid-liquid extraction with the solvent ethyl acetate, subsequently chromatography: gel-filtration (Sephadex G-75) and ion exchange (SP-Sepharose and DEAE-cellulose). The antimicrobial in question did not adhere to the SP-Sepharose column, showing that it has negative charge. The antimicrobial activity of the compound was recovered after each step. The compound remained active after the stability tests like the addition of EDTA, proteolytic enzymes and high temperatures. This suggests that the antimicrobial compound is not a protein. This work suggests further studies based on the obtained preliminary results such as the amplification of the entire 16S rDNA of actinobacterias isolated fragment and the investigation of the antimicrobial compound using high resolution chromatography.

Made available in DSpace on 2016-06-02T20:36:18Z (GMT). No. of bitstreams: 1 2209.pdf: 3433970 bytes, checksum: f1696bf3d4dba44ef3cb8eb1b129cdf5 (MD5) Previous issue date: 2008-10-11 / Financiadora de Estudos e Projetos / This work describes the investigation of part of the secondary metabolism of actinomicete Streptomyces clavuligerus and endophytic filamentous fungus Penicillium sp isolated from Murraya paniculata; Penicillium brasilianum and Aspergillus aculeatus isolated from Melia azedarach and Penicillium griseoroseum isolated from the grains of Coffea arabica. Preliminary tests were realized to define the best composition of medium culture and the best condition of cultivation Streptomyces clavuligerus. On the other hand, the endophytic fungus were cultivated in rice and Czapec´k (medium liquid). The anthranilic acid and the 7-hydroxy-2- methyl-chromone were identified from the extracts of S. clavuligerus by spectroscopic methods 1D e 2D NMR and mass spectrometry. Methodologies of analysis for detection of β-lactam antibiotics were developed by liquid chromatography combined with mass spectrometry. The identification was based on studies of standard compounds and comparison with literature data. These methodologies were efficient in the analysis of this type of substances and they allowed the detection and identification of β-lactam antibiotics such as the penicillin N, the cephamycin C, deacetoxy-cephalosporin C and the clavulanic acid in small amounts in S. clavuligerus the extracts. In the endophytic fungus extracts were possible to identify the 6-aminopenicillanic acid, precursor of the penicillin G and metabolites no β-lactams as nucleotides thymine and adenine. / Este trabalho descreve a investigação química de uma parte do metabolismo secundário do actinomiceto Streptomyces clavuligerus e dos fungos endofíticos filamentosos Penicillium s.p isolado de Murraya paniculata; Penicillium brasilianum e Aspergillus aculeatus isolados de Melia azedarach e Penicillium griseoroseum isolado dos grãos de Coffea arabica. Inicialmente, foram realizados testes preliminares para definir a melhor composição do meio de cultura e as melhores condições de cultivo para Streptomyces clavuligerus, já os fungos endofíticos foram cultivados em arroz e em meio líquido Czapec k. Dos extratos de S. clavuligerus foram identificados, por métodos espectroscópicos de RMN 1D e 2D e espectrometria de massas, o ácido antranílico e a 7-hidroxi-2-metilcromona. Com o objetivo de determinar a presença de antibióticos β-lactâmicos e outros metabólitos nos extratos foram desenvolvidas metodologias de detecção por cromatografia líquida acoplada a espectrometria de massas. A identificação das substâncias foi feita com base no estudo de padrões comerciais e levantamento bibliográfico. Estas metodologias se mostraram eficientes na análise deste tipo de substâncias e permitiu a detecção de antibióticos β-lactâmicos como a penicilina N, a cefamicina C, a deacetoxicefalosporina C e o ácido clavulânico nos extratos de S. clavuligerus. Estas metodologias foram aplicadas nas análises dos extratos fúngicos onde foi identificado o ácido 6-aminopenicilânico, precursor da penicilina G e metabólitos não β-lactâmicos como os nucleotídeos timina e adenosina.

Para determinar as variáveis morfogênicas, estruturais e o fluxo de tecidos os tratamentos foram duas intensidades (baixa e moderada) e dois métodos de pastejo (pastejo com lotação contínua e rotacionada). No experimento 1 o bastão graduado apresentou a melhor correlação com a massa de forragem (r2=0,65). No 2 as melhores correlações foram obtidas quando avaliadas as faixas de pós-pastejo para o disco medidor (r2=0,47) e as de pré-pastejo para o bastão graduado (r2=0,36). Para as variáveis morfogênicas e estruturais as intensidades de pastejo foram responsáveis por diferenças na taxa de elongação de folhas (intensidade baixa resultou em maior taxa de elongação) e nas características estruturais (intensidade baixa resultou em menor densidade de perfilhos, maior comprimento e número de folhas vivas). Os métodos de pastejo influenciaram as características morfogênicas (lotação contínua resultou em maior taxa de elongação de folhas, maior taxa de surgimento e tempo de vida das folhas no ciclo de observação I) e estruturais (lotação contínua resultou em maior densidade de perfilhos); bem como foi obtida interação com as intensidades e com os ciclos de avaliação. O fluxo de crescimento (favorecido por lotação rotacionada a baixa intensidade) e de senescência (favorecido por lotação contínua a baixa intensidade) foram afetados pelos tratamentos, enquanto que o fluxo de consumo não foi alterado pelos tratamentos.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-08-25Bitstream added on 2014-06-13T18:31:30Z : No. of bitstreams: 1 oliveira_gm_me_rcla.pdf: 243960 bytes, checksum: a79bd4d2995d8e646a1a343bffbaba1d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Neste trabalho estudou-se a queratinase produzida por uma cepa de Streptomyces sp LMI-1 isolada de uma fábrica de processamento de aves domésticas. A enzima degradou 87% das penas após 120h de cultivo. Ela foi estimulada por íons Ba+2 e inibida por Ca+2, Mn+2, EDTA e Hg+. O pH ótimo de atividade da enzima foi 8,5 e a temperatura de maior atividade foi 60oC. A enzima é estável por até 2h em 50oC. Na análise do caldo de cultura detectou-se a presença dos aminoácidos serina, metionina, prolina, tirosina e leucina logo após 72h de cultivo. A enzima apresenta potencial para utilização industrial na produção de ração animal. / In the present work was studied keratinase produced by Streptomyces sp LMI-1 isolated of an industrial plant of poultry processing when cultived with feathers as a carbon source. The enzyme degraded 87% of feathers after 120 hours. The enzyme was stimulated by Ba+2 and inhibited by Ca+2, Mn+2, EDTA and Hg+. The optimum pH and temperature for the enzyme was 8,5 and 60oC, respectively. The enzyme was stable after 2 hours at 50oC. The culture broth analysis by thin layer chromatogram showed presence of amino acids serine, methionine, proline, tyrosine and leucine after 72 hours of incubation. The enzyme presents potential for industrial use in the production of animal feed preparation.

Made available in DSpace on 2014-06-11T19:23:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-12-12Bitstream added on 2014-06-13T19:08:57Z : No. of bitstreams: 1 antonio_t_me_araiq.pdf: 609701 bytes, checksum: da17caf99f88297148973c0dd3d64e83 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os grupos de antibióticos mais importantes clinicamente são os dos b-lactâmicos, aminoglicosídeos e tetraciclinas. Streptomyces clavuligerus produz vários compostos b- lactâmicos, com destaque para os antibióticos envolvidos na rota biossintética da cefalosporina C (penicilina N, deacetoxicefalosporina C e cefamicina C) e o ácido clavulânico (AC) que, embora não tenha atividade biológica significativa, é um potente inibidor de b-lactamases (penicilinases e cefalosporinases). A cefamicina C (CefC) é uma 7-metoxi-cefalosporina que apresenta maior atividade que a cefalosporina C (CPC), produzida somente por fungos, por ser resistente a b-lactamases. Apesar das rotas biossintéticas de AC e CefC serem completamente independentes em S. clavuligerus, são controladas pelo mesmo elemento multifuncional (ccaR), o que dificulta a indução da produção de um ou outro composto durante o processo fermentativo. No presente trabalho, procurou-se obter maiores concentrações de CefC manipulando-se componentes em meio solúvel de cultivo de S. clavuligerus, selecionados dentre compostos que, segundo a literatura, atuam como agentes reguladores da síntese daquele antibiótico. As fermentações foram realizadas em frascos agitados (28ºC, 260 rpm) para selecionar fontes de C e de N e, então, avaliar o processo no melhor meio-padrão, variando-se concentrações combinadas de L-lisina (10 a 108 mM) e -cetoglutarato (3 a 110 mM) através de metodologia de planejamento experimental. A presença de -cetoglutarato acarretou em aumento indesejável de pH, afetando negativamente o processo e os melhores resultados (entre 300 e 400 mg CPC totais/L após 72 horas de fermentação) foram obtidos no meio adotado como meio-controle, contendo amido e extrato protéico de semente de algodão como principais fontes de C e N, respectivamente, e L-lisina. / The most important groups of antibiotics, from a clinical standpoint, are -lactams, aminoglycosides and tetracyclines. Streptomyces clavuligerus produces several -lactam compounds, primarily the antibiotics involved in the biosynthetic route of cephalosporin C (penicillin N, deacetoxycephalosporin C and cephamycin C) and clavulanic acid (CA), which, despite its slight biological activity, is a potent inhibitor of -lactamases (penicillinases and cephalosporinases). Cephamycin C (CMC) is a 7-methoxycephalosporin with higher bioactivity than cephalosporin C (CPC) because it is more resistant to -lactamases. Although the biosynthetic routes of CA and CMC are completely independent in S. clavuligerus, they are controlled by a common multi-functional element (ccaR), which hinders induction of the production of one or the other compound during the fermentation process. In this work, we sought to obtain higher concentrations of CMC by handling compounds in a soluble medium of S. clavuligerus, which were selected from compounds that, according to the literature, act as regulating agents in the synthesis of that antibiotic. Fermentation was carried out in flasks under shaking (28ºC, 260 rpm), in order to select sources of C and N. The process was then evaluated in the best standard medium, by varying combined concentrations of lysine (10 to 108 mM) and -ketoglutarate (3 to 110 mM) using an experimental planning methodology. The presence of -ketoglutarate caused an undesirable increase in pH, negatively affecting the process. The best results (between 300 and 400 mg/L of total CPC after 72 h of fermentation) were obtained in the medium used as the control, which contained starch and cottonseed protein extract as main sources, respectively, of C and N, and L-lysine.