Two fungal pathogen species, Helminthosporium solani and Pythium ultimum, cause significant economic loss to potato (Solanum tuberosum) growers throughout the world. These pathogens have substantial differences in cellular makeup, pathogenicity, and modes of infection. We studied the efficacy of 82 isolates within the bacterial genus Streptomyces in inhibiting these pathogens under laboratory and greenhouse conditions. Derivatives of Streptomyces have significant implications in medicinal use because of their antibiotic and antifungal properties. Under in-vitro conditions, 25% of Streptomyces isolates inhibited growth of P. ultimum, up to 81%. Ninety-five percent of the Streptomyces isolates inhibited growth of H. solani, with a maximum of 70%. In storage, these findings lead us to believe substantial differences between Streptomyces isolates will allow for some isolates to be effective biological controls at controlling diseases on common pathogens of potatoes.

Bacteriophages, or simply "phages," are the most abundant biological entities on the planet and are thought to be the largest untapped reservoir of available genetic information. They are also important contributors to both soil health and nutrient recycling and have significantly influenced our current understanding of molecular biology. Bacteria in the genus Streptomyces are also known to be important contributors to soil health, as well as producing a number of useful antibiotics. The genetic diversity of large (> 30) groups of other actinobacteriophages, i.e. phages infecting a few close relatives of the Streptomycetes, has been explored, but this is the first formal effort for Streptomyces-infecting phages. Described here are a group of 45 phages, isolated from soil using a single Streptomycete host, Streptomyces griseus ATCC 10137. All 45 phages are tailed phages with double-stranded DNA. Siphoviruses predominate, six of the phages are podoviruses, and no myoviruses were observed. Notably present are seven phages with prolate icosahedral capsids. Genome lengths and genome termini vary considerably, and the distributions of each are in line with findings among other groups of studied actinobacteriophages. Interestingly, the average G+C among the 45 phages is around 11% lower than that of the isolation host, a larger disparity than reported for other groups of actinobacteriophages. Eighteen of the phages carry between 17 and 45 tRNAs and 12 of those carry a single tmRNA. Forty-three phages were grouped into seven clusters and two subclusters based on dot plot analysis, average nucleotide identities, and gene content similarities. Two phages were not clustered with other phages in this dataset. A total of 5250 predicted genes were sorted into 1300 gene "phamilies," with about 8% of the total phamilies having only a single member. Analysis of gene content among the 45 phages indicates first that most clusters presented here appear to be relatively isolated from one another, with phages in any one cluster generally sharing < 10% of their genes with phages in other clusters described here. Secondly, most of the phages here are more than twice as likely to share genes with phages isolated on bacteria outside of the genus Streptomyces than they are other phages isolated using a Streptomycete as host. These observations suggest that (1) the phage clusters here have a distinct extended host range, (2) those host ranges share overlap, and (3) Streptomyces griseus is likely not the preferred natural host for all phages described.

Orientador: Fernando Lucas Primo / Resumo: A exploração da cor nunca esteve tão evidente como nos dias de hoje, e muitas indústrias são direta ou indiretamente dependentes da disponibilidade de corantes artificiais. Com o crescimento da produção, consumo e utilização de produtos contendo corantes sintéticos, aumentaram também os relatos com problemas relacionados à saúde e aos danos causados no meio ambiente devido, principalmente a sua baixa biodegradabilidade, fazendo-se repensar o uso dessas substâncias. Frente ao conhecimento do público, assim como a relação à segurança ambiental e a preocupação com a saúde, os corantes naturais nesse sentido podem ser uma alternativa ao uso dos corantes sintéticos conquistando, a cada ano, uma nova fatia do mercado. O objetivo deste trabalho foi caracterizar o corante verde de origem biotecnológica, obtido a partir do cultivo do microrganismo Streptomyces carpaticus, através de diversas técnicas espectroscópicas, para que assim possa se propor a aplicação do mesmo em indústrias têxteis, de cosméticos ou alimentícias. Foram realizadas análises para identificação do corante bem como testes para definição dos solventes de trabalho, os quais foram utilizados na preparação das amostras que foram enviadas para as diversas técnicas. Os testes iniciais realizados no corante bruto, tiveram por objetivo definir o solvente ideal para preparação das amostras para análises de Espectroscopia de UV-Vis, Fluorescência, Espectrometria de massas e Ressonância Magnética Nuclear uni e bidimensionais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Color exploration has never been more evident than it is today. Many industries are directly or indirectly dependent on the availability of synthetic dyes. With the growth of the production, consumption and the use of products containing synthetic dyes the number of reports about problems related to health and damage caused to the environment, mainly due to their low biodegradability have been increasing. So the use of these substance should be rethought. In the face of public knowledge, as well as environmental safety and health concerns, natural dyes in this sense can be an alternative to the use of synthetic dyes, gaining a new market share each year. The objective of this work was to characterize the green dye of biotechnological origin, obtained from the cultivation of the microorganism Streptomyces carpaticus, through several spectroscopic techniques, so that it can be proposed its application in the textile, cosmetic or food industries. Analyzes were performed to identify the dye as well as tests to define the working solvents, which were used in the preparation of samples that were sent to the various techniques. The initial tests carried out on the raw dye aimed to define the ideal solvent for sample preparation for analysis of UV-Vis Spectroscopy, Fluorescence, Mass Spectrometry and One and Two Dimensional Nuclear Magnetic Resonance. After Infrared and NMR analysis, it was possible to verify the presence of several contaminants, which directed the work to the dy... (Complete abstract click electronic access below) / Mestre

No description available.

In Streptomyces bacteria, the expression of many antibiotic biosynthetic clusters is controlled by both cluster-specific regulators and more globally-acting regulators; however, much remains unknown about the factors that govern antibiotic production. In Streptomyces venezuelae, we have discovered that the broadly-conserved nucleoid-associated protein Lsr2, plays a major role in repressing specialized metabolic cluster gene expression. To understand how Lsr2 exerts its gene silencing effects, we focused our attention on the well-studied, but transcriptionally silent, chloramphenicol cluster in S. venezuelae. We established that Lsr2 represses transcription of the chloramphenicol cluster by binding DNA both within the cluster and at distal positions. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not under Lsr2 control. We discovered that CmlR functions to ‘counter-silence’ Lsr2 activity, alleviating Lsr2 repression and permitting chloramphenicol production, by recruiting RNA polymerase. Lsr2 plays a central role in controlling antibiotic production in Streptomyces; however, beyond this counter-silencing activity, little is known about how Lsr2 is regulated. We identified regulators that could control the expression of lsr2, and found that Lsr2 and LsrL, an Lsr2 homologue that is encoded by all streptomycetes, interact directly with each other, and that their respective DNA-binding activities are altered by the presence of the other protein. These data suggest that LsrL may impact Lsr2 activity in regulating antibiotic production in Streptomyces. Beyond Lsr2, we wanted to develop a comprehensive understanding of the regulatory proteins that impact biosynthetic gene cluster expression. To define the regulatory protein occupancy of antibiotic clusters, we developed ‘in vivo protein occupancy display-high resolution’ (IPOD-HR) technology for use in Streptomyces. This work will lay the foundation for establishing a comprehensive regulatory network map for biosynthetic clusters in Streptomyces, and guide future work aimed at stimulating the expression of metabolic clusters in any Streptomyces species. / Thesis / Doctor of Philosophy (PhD) / Streptomyces bacteria produce the majority of naturally-derived antibiotics, and they have the genetic potential to produce many more antibiotics and antibiotic-like compounds (‘specialized metabolites’). Specialized metabolism is controlled by multiple regulatory systems. In Streptomyces venezuelae, we have discovered that the nucleoid-associated protein, Lsr2, represses the expression of most specialized metabolic clusters, and manipulating Lsr2 activity can stimulate antibiotic production. To better understand how Lsr2 exerts its repressive effect, we explored how Lsr2 controlled the production of a known antibiotic. We ultimately identified multiple regulators that could impact the expression and/or activity of Lsr2. Building on the regulatory foundation provided by Lsr2, we then set out to establish a comprehensive regulatory network that governs biosynthetic gene cluster expression. Collectively, this work improves our understanding of antibiotic gene regulation in Streptomyces bacteria, and has the potential to guide novel strategies aimed at stimulating the production of new antibiotics in Streptomyces.

Earlier investigations indicated that the genes involved in the biosynthesis of tryptophan from anthranilic acid were located in at least two separate regions on the chromosome of Streptomyces coelicolor A3(2). Tryptophan-requiring mutants capable of utilizing indole mapped counterclockwise of hisC9 between hisC9 and proAl. Trp mutants unable to use indole in place of tryptophan mapped clockwise of hisC9 between hisC9 and ammA5. Other genetic markers, located between proAl and hisC9, include leuAl, rif'B37 and thiC2. Twenty indole-utilizing Trp mutants were mapped relative to leuAl, rifB37 and thiC2. All but two recently isolated mutants, Trp-e8 and e22, mapped between rifB37 and thiC2. Genetic and enzymatic analyses of Trp-e8 and e22 indicate that these mutants map between hisC9 and ammA5, fail to complement each other in vivo and lack indoleglycerol phosphate synthase. Two mutants, Trp-e14 and 8, mapping between hisC9 and ammA5 were assayed for their tryptophan enzymes. Trp-el4 lacks tryptophan synthase B activity. Tryptophan synthase A activity was missing and tryptophan synthase B activity was relatively weak in crude extracts of Trp-8. Anthranilate-PRPP phosphoribosyltransferase activity was not detected in crude extracts of two mutants, Trp-e6 and e10, mapping between rifB37 and thiC2. / Master of Science

A antracnose da soja é uma doença que causa danos na cultura, principalmente na fase vegetativa. Devido aos danos causados pela doença, métodos de controle integrado estão sendo estudados. Um desses métodos é o controle biológico, no qual agentes bióticos ou seus metabólitos são usados para controlar o patógeno. Esses agentes podem atuar diretamente no patógeno ou como indutores de resistência nas plantas e, assim, contribuir para o desenvolvimento de produtos biológicos. A bactéria Streptomyces scabies produz uma toxina que pode induzir resistência em plantas, a taxtomina A. O objetivo desta pesquisa foi avaliar os efeitos diretos e indiretos da taxtomina parcialmente purificada (TPP) no controle de Colletotrichum truncatum (C. truncatum). O efeito direto da TPP na germinação de esporos e no crescimento micelial do fungo foi testado in vitro. Nos testes in vivo foram realizados a padronização do método de inoculação de C. truncatum, a escolha da cultivar a partir da reação de cultivares e o efeito de indutores na resistência sistêmica e local. Análises bioquímicas da atividade da peroxidase (POX) e teor de fenóis livres e ligados também foram conduzidos. Como resultado, observou-se que a TPP autoclavada ou não teve efeito inibitório na germinação de esporos de C. truncatum Por outro lado, a TPP não afetou o crescimento micelial do patógeno. O melhor método para inoculação de C. truncatum em soja foi via micelial, com 72 h de umidade foliar e utilizando-se o isolado CMES 1059. No teste de reação das cultivares, a DS5916IPRO foi uma das cultivares mais suscetíveis dentre as 16 testadas. No tocante a indução de resistência local, as cultivares tratadas com TPP, Bion (Acibenzolar - S - Metil) e o fungicida Priori Xtra&#174; (azostrobina+citroconazol) apresentaram a menor severidade entre os tratamentos testados. Na avaliação da indução de resistência sistêmica, os tratamentos não diferiram (p> 0,05) do controle. As análises bioquímicas também não diferiram. Assim, pode-se concluir que a TPP na dose de 100 &#956;g equivalentes de taxtomina/L é termoestável e tem efeito direto sob a germinação de conídios de C. truncatum. Além disso, a TPP e o ASM têm o potencial de induzir resistência local em plantas de soja contra o patógeno. / The anthracnose of soybean is a disease that causes severe damages in the crop, mainly in the vegetative phase. Due to disease damage, methods of integrated control has been studied. One of these methods is the biological control, in which a biotic agent or its metabolites are used to control a pathogen. These agents can act directly on the pathogen and/or act as resistance inducers in plants and thus contribute to the development of biological products. The bacterium Streptomyces scabies produces a toxin called thaxtomin that can induce resistance in plants. The objective of this research was to evaluate the effects of the partially purified thaxtomin (TPP) on the activation of biochemical defense mechanism in the soybean plants. Thus, the direct effect of TPP on spore germination and mycelial growth was tested in vitro. The in vivo test included standardization of inoculation of C. truncatum, cultivars reaction and the effect of inducers in the systemic and local resistance. Biochemical analyzes of peroxidase activity (POX) and content of free and bound phenols were also performed. It was observed that autoclaved or non-autoclaved TPP exhibited inhibitory effects on the germination of C. truncatum. On the other hand, TPP did not affect the mycelial growth of the pathogen. The best method for inoculation of C. truncatum was by using mycelium, with 72 h of leaf wetness and with the isolate CMES 1059. In the cultivar reaction test, it was determinate that the cultivar DS5916RR was the most susceptible out among the 16 cultivars tested. Regarding local induction resistance, the cultivars treated with TPP, Acibenzolar - S - Methyl (ASM) and fungicide Priori Xtra® exhibited the lowest severity among the treatments tested. In the evaluation of systemic resistance induction, the treatments didn\'t differ (p> 0.05) of the control. The biochemical analyses also didn\'t differ among then. Thus, it can be concluded that TPP is thermostable and has direct effect on conidium germination of C. truncatum. Besides that, the TPP and ASM have the potential to induce local resistance in soybean plants against the pathogen.

Ácido clavulânico (AC) é um potente inibidor de &beta;-lactamases, produzido por Streptomyces clavuligerus, usado clinicamente em combinação com antibióticos &beta;-lactâmicos para tratar infecções bacterianas resistentes. Apesar da produção industrial de AC já ser bem estabelecida muitos aspectos importantes relacionados com sua biossíntese permanecem carentes de estudo. Sabe-se que a via de síntese do AC envolve no mínimo 8 passos enzimáticos sendo os primeiros passos mais abordados. Por exemplo, as enzimas N2-(2-carboxietil) arginina sintase (CEAS), &beta;-lactama sintase (BLS) e proclavaminato amidino hidrolase (PAH) são as responsáveis pela primeira, segunda e quarta reações enzimáticas respectivamente. Estudos mutagênicos recentes em S.clavuligerus relacionaram cópias extras dos genes ceas, bls e pah (ceas1, bls1 e pah1) com essa via porém nenhum ensaio enzimático foi relatado. Embora os passos finais da via ainda não estejam completamente estabelecidos, a ação de algumas enzimas putativas, como a codificada por orf12, mostraram ser essenciais a produção do AC. Assim, com o objetivo de aumentar a informação disponível sobre a biossíntese do AC estudamos quatro de seus membros: CEAS1, BLS1, PAH1 e a proteína putativa codificada pela orf12. Os genes foram isolados a partir do DNA genômico de S. clavuligerus por PCR e clonados em vetores para produção das proteínas recombinantes em E.coli. Os protocolos de expressão foram estabelecidos para CEAS1, PAH1 e ORF12 e as proteínas recombinantes foram purificadas por cromatografia de afinidade por metal. BLS foi obtida de forma isolúvel. As proteínas solúveis foram caracterizadas por meio de técnicas bioquímicas e estruturais. As análises de CEAS1 e PAH1 foram comparadas com informações já obtidas para as isozimas CEAS2 e PAH2, respectivamente. Assim, as análises de oligomerização das proteínas resultaram em uma mistura de oligômeros (monômero, dímero e tetrâmero) para CEAS1, na forma hexamérica para PAH1 e na forma dimérica para ORF12, estando de acordo com as formas solúvel e cristalográfica de CEAS2 (dímero e tetrâmero) e PAH2 (hexâmero). Espectros de dicroísmo circular mostraram que CEAS1 e PAH1 possuem um enovelamento do tipo &alpha;/&beta; sendo estáveis até 35ºC e numa ampla faixa de pH. Os parâmetros termodinâmicos da interação entre CEAS1 e o cofator Mg+2 foram determinados mostrando que é entropicamente dirigida, com uma estequiometria de ligação de 4 : 1, com uma constante de afinidade na ordem de micromolar (KD = 1,76 &plusmn; 0.23 &micro;M). Análises realizadas com as técnicas de reação acoplada, de Cromatografia Líquida de Alta Pressão acoplada a Espectrometria de Massas (LC-MS) e de Calorimetria de Titulação Isotérmica mostraram que CEAS1, assim como CEAS2, apresenta atividade sob o substrato gliceraldeído-3-fosfato, porém sem a formação do produto final N2-(2-carboxietil)arginina. Por outro lado, a proteína recombinante PAH1 mostrou ser inativa sobre o substrato análogo, N-&alpha;-acetil-L-arginina. Assim, apesar das isozimas manterem um padrão estrutural, podem ter mecanismos de ação distintos. Em relação a ORF12 esta proteína foi classificada com uma &beta;-lactamase com atividade esterase de acordo com nossos estudos realizados com os substratos cefalosporina C e p-nitrofenil acetato. / Clavulanic acid (CA) is a potent inhibitor of &beta;-lactamases, produced by Streptomyces clavuligerus, clinically used in combination with &beta;-lactam antibiotics to treat resistant bacterial infections. Although CA industrial production is well-established, many important aspects related to its biosynthesis remains under study. It is known that CA pathway involves at least 8 enzymatic steps, being the earliest stages more addressed. For instance, N2-(2-carboxyethyl) arginine synthase (CEAS), &beta;-lactam synthase (BLS) and proclavaminate amidinohydrolase (PAH) are responsible for the first, second and fourth enzymatic reaction, respectively. Recent mutagenic studies in S.clavuligerus have related extra copies of ceas, bls and pah genes ((ceas1, bls1 e pah1) to this pathway but none enzymatic assay was further reported. Although later stages the pathway remain unclear, the action of some putative enzymes like the codified by orf12 showed essential to CA production. Thus, aiming to increase the information available about CA biosynthesis we studied four of its members: CEAS1, BLS1, PAH1, and the putative protein codified by orf12. The genes were isolated from S.clavuligerus genomic DNA by PCR and further cloned into expression vectors in order to produce recombinant proteins in E.coli. Protocols of protein expression were established to CEAS1, PAH1 and ORF12 and recombinant proteins were purified by metal affinity chromatography. BLS was obtained as an insoluble form. Soluble proteins were characterized by means of biochemical and structural approaches. Analyses of CEAS1 and PAH1 were compared with information ever conducted to the isozymes CEAS2 and PAH2, respectively. Thus, oligomerization analysis of proteins resulted respectively in a mix of oligomers forms (monomer, dimer, tetramer) to CEAS1, hexameric form to PAH1 and dimeric form to ORF12, according to the soluble and crystallographic form of CEAS2 (dimer and tetramer) and PAH2 (hexamer). Circular Dichroism spectra showed that CEAS1 and PAH1 have an &alpha;-&beta; conformation and were stable up to 35ºC over a wide pH range. Thermodynamic parameters of CEAS1 cofactor (Mg+2) binding were determined showing that is entropic driven, with a 4:1 binding stoichiometry, with a micro-molar affinity (KD = 1.76 &plusmn; 0.23 &micro;M). Analyses by coupled assay, High Pressure Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and Isothermal Titration Calorimetry showed that CEAS1, as well as the CEAS2, presents activity at the substrate glyceraldehydes-3-phosphate, however without formation of final product, N2-(2-carboxyethyl)arginine. Meanwhile recombinant PAH1 showed none activity at analogous substrate, N-&alpha;-acetil-L-arginine. Thus despite isozymes maintain a structural pattern, they may have distinct action mechanism. Regards to ORF12, this protein was classified as a &beta;-lactamase with an esterase activity according to our studies performed with the substrates cephalosporin C and p-nitrophenyl acetate.

Les bactéries du genre Streptomyces produisent de nombreux métabolites secondaires, dont certains possèdent des propriétés intéressantes en agriculture et en pharmaceutique. Avec le développement de la génomique, de nombreux outils bioinformatiques de recherche de groupes de gènes du métabolisme secondaire ont été développés au cours de la dernière décennie pour explorer les génomes. Ces outils sont basés sur la recherche de similarité de séquences et de ce fait, les clusters atypiques, constitués de gènes non caractérisés, ne peuvent être détectés par ces approches. L'isolement de tels clusters nécessite donc la mise en œuvre de nouvelles stratégies. La comparaison d’espèces d'Actinomycetes proches a révélé que les îlots génomiques, régions présentes dans un seul génome, sont très souvent enrichis en gènes du métabolisme secondaire. Nous avons participé (en collaboration avec les équipes d’Olivier Lespinet et de Pierre Leblond et Bertrand Aigle) au développement d’un outil, Break Viewer, permettant de localiser les îlots génomiques en comparant des génomes proches de Streptomyces. Cet outil a permis l'identification d'un îlot non détecté par les approches classiques, îlot dont l'étude a montré qu'il contenait un groupe de gènes du métabolisme secondaire. L’étude de ce groupe de gènes a montré qu'il dirige la synthèse de trois composés, le produit majoritaire étant le sphydrofurane. Une analyse fonctionnelle du cluster sphydrofurane a permis de déterminer les gènes impliqués dans la biosynthèse et la régulation de la biosynthèse du sphydrofurane et de proposer un modèle préliminaire pour la biosynthèse de ce métabolite. / Streptomyces are soil-dwelling bacteria that produce numerous secondary metabolites, some of which have interesting properties in agriculture and pharmaceuticals. With the development of genomics, many bioinformatics tools to search genomes for secondary metabolism gene clusters have been developed over the last decade. These tools are based on sequence similarity searches and therefore atypical clusters, consisting of uncharacterized genes, cannot be detected by these approaches. The isolation of such atypical clusters therefore requires the implementation of new strategies.Comparing closely related Actinomycetes species revealed that genomic islands (regions that are present in one genome only), are often enriched in secondary metabolite genes. We participated (in collaboration with the team of Olivier Lespinet and the team of Pierre Leblond and Bertrand Aigle) to the development of a new tool, Break Viewer, to locate genomic islands by comparing the genomes of closely related Streptomyces. This tool allowed the identification of an island, undetected by conventional approaches, island whose study showed that it contained a secondary metabolism gene cluster. The study of this cluster has shown that it directs the synthesis of three compounds, the major product being sphydrofuran. A functional analysis of the sphydrofuran gene cluster allowed us to identify the genes involved in the biosynthesis and regulation of sphydrofuran and to propose a preliminary model for the biosynthesis of this metabolite.

Encapsulation is a novel technique that involves the entrapment of materials such as cells, enzymes or chemicals within a semi-permeable matrix and is being explored as a drug delivery system. This project investigated the encapsulation of Streptomyces nodosus in alginate to assess whether this organism can produce the antifungal drug amphotericin B from within the matrix. New methods were developed to immobilise S. nodosus mycelia and spores in alginate capsules, assess bacterial viability and detect ng mL–1 quantities of amphotericin B in culture fluids. When capsules were cultured and cell proliferation was encouraged, organisms formed protrusions on the surface of the capsules. Differentiated branched hyphae that never progressed to sporogenic hyphae were observed on the surface of these structures. Viability was maintained for up to 30 days and low levels of amphotericin B were produced. The emergence of a co-existing free-dwelling population was also observed. Culturing immobilised organisms using conditioned media from an amphotericin deficient S. nodosus strain, augmented the development of the free-dwelling population resulting in the detection of amphotericin B in the culture fluid and full differentiation to sporogenic hyphae. This is the first report of sporulation of S. nodosus in liquid environments and demonstrates that immobilised S. nodosus can produce antibiotics. The sporulation of free-dwelling organisms was also induced using conditioned media and manipulation of quorum size, indicating a solid surface is not required for sporulation. Conditioned media from other Streptomyces spp. induced variable responses including sporulation, pigment formation and antibiotic production, possibly demonstrating communication between species and/or alteration in nutritional status. This new model for the life cycle of S. nodosus will permit the study of developmental pathways, antibiotic production, microbial community structure and inter-species and intra-species signalling. / Doctor of Philosophy (PhD)