As antimicrobial resistance persistently disrupts the treatment of microbial infection, natural product chemistry has played a significant role in identifying novel drugs with novel modes of action critical to getting ahead of resistance. The primary goal of this project is to extract and identify potential novel antimicrobial natural products produced by Arthrobacter sp. Although underexplored, Arthrobacter sps. have been known to produce bioactive compounds with versatility; one such is a depsipeptide with quorum-sensing inhibitory activity.1 In this research, an Arthrobacter sp. TAJX1902, isolated from a soil sample, showed inhibitory activity against a filamentous indicator bacterium and a violacein-producing Janthinobacterium sp. Arthrobacter sp. TAJX1902 was cultured using rich medium broth and agar to extract and isolate metabolites. Isolated compounds were characterized via spectroscopic techniques, including 1D and 2D-NMR spectroscopy and GCMS analysis. Arthrobacter sp. TAJX1902 produces Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl), and five other bioactive cyclic dipeptides (CDPs).

Over the past century, dust emissions have increased in frequency and intensity due to anthropogenic influences and extended droughts. Dust transports microbes, nutrients, heavy metals and other materials that may then change the biogeochemistry of the receiving environments. The purpose of this study was to find whether unique bacterial communities may provide distinct fingerprints of dust sources in the Western USA. We collaborated with the National Wind Erosion Research Network (NWERN) to identify bacterial core communities (core) of dust from ten NWERN sites, and compared communities to location, soil, and regional characteristics. In order of importance, precipitation levels (F = 43, P = 0.0001, Df = 2, r2 = 0.25), location (F = 16, P = 0.0001, Df = 5, r2 = 0.23), soil texture (F = 14, P = 0.0001, Df = 3, r2 =0.12), seasonality (F = 11, P = 0.0001, Df = 2, r2 = 0.064), and elevation (F = 5.7, P = 0.0002, r2 = 0.033) determined bacterial community composition. Bacterial core communities were defined as taxa present in at least 50% of samples at each site and offered predictable patterns of dust communities in terms of abundant (> 1% relative abundance) and rare (< 1% relative abundance) signatures. We found distinct bacterial core communities that reflected dust source systems, for example, sites contaminated with heavy metals contained Romboutsia, Turicibacter, Clostridium sensu stricto 1, Geodermatophilus, and Microvirga. Sites with association to plants and biocrusts contained Methylobacterium-Methylorubrum, Bradyrhizobium, Paenibacillus thermoaerophilus, Cohnella, and bacterial families Solirubrobacteraceae, Sphingobacteraceae, and Myxococcaceae. The presence of Sphingomonas, Stenotrophomonas, Rhodococcus, and Phenylobacterium were found in hydrocarbon contaminated soils. High stress (UV radiation and desiccation) sites contained Deinococcus, Blastococcus, and Modestobacter. We found that seasonal changes affected microbial community composition in five NWERN sites (CPER, HAFB, Jornada, Red Hills, and Twin Valley) (p < 0.05), while no seasonal effects on bacterial distribution were observed at Moab. Our results identify that the use of core microbiomes may offer a fingerprinting method to identify dust source regions.

<p>  </p> <p>Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated, with many natural products being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. These elicitors can be natural products, including antibiotics or hormones, or synthetic compounds. The use of small molecule elicitors to induce the production of secondary metabolites has several advantages. First, addition of elicitors to fermentation media can result in increased titers of known natural products. Second, elicitors can enable the discovery of novel natural products typically produced at undetectable levels. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of “silent” natural products. In this work, we sought to expand on the method of chemical induction by utilizing oxytetracycline at a sub-MIC concentration to induce secondary metabolite production in Streptomyces. We have shown that translation-inhibiting antibiotics, specifically oxytetracycline, have a profound effect on the production of coeliomycin P1, actinorhodin, and calcium-dependent antibiotics (CDAs) in S. coelicolor and S. lividans. The expression of actinorhodin in S. lividans under these conditions is unique, unlike its counterpart, S. coelicolor, which can produce actinorhodin under standard conditions. In addition to the increased production of known secondary metabolites, we have also demonstrated the induction of BGCs in several other strains of Streptomyces, which were observed via LC-MS. </p> <p>In addition to exploring antibiotics as elicitors we have explored the traditional approach of natural product discovery by taking an bioactivity guided approach. Several strain that we isolated from soil collect of Hawaii were screened for activity against several pathogenic strains primarily looking for which strain will inhibit the growth of a. baumannii, which is an intriguing target because the rate of resistance to common antibacterial medication is rising and it’s membrane composition is vastly different compared to other gram negative bacterium like E.coli. From this preliminary screening 1 strain (Streptomyces sp. CS62) out of the 8 that tested exhibited the desired biological activity. The supernatant of Streptomyces sp .CS62 was processed and screen by LC-MS to gain insight on the type of molecules that Streptomyces CS62 could produce. Upon our initial screening process none of the masses observed in the mass spec were matched to knowns. However, after 2D NMR analysis and genomic analysis it was unveiled that Streptomyces sp. CS62 produces factumycin a known antibacterial agent that targets A.baumannii .This unfourtunate turn of events illustrates the issues with natural product discovery and the need to improve natural product databases.</p> <p>In conjunction to discovering a novel producer of factumycin we are also investigating the production of antifungal compounds from Staphylococcus lugdunensis  a commensal strain that modulates the microbiome by producing lugdunin. The supernatant collected of Staphylococcus lugdunensis  is exclusively being test against Candida auris due to the immense health risk it possess to society because of its innate resistance to many antifungal drugs and its ability to rapidly gain resistance to other classes of antifungals.</p> <p>In addition to exploring the influence of antibiotics on secondary metabolite production and using bioactivity as a guide to discovering antibiotics. We are evaluating the soils collected from unique environments as potential sources for novel natural products. Specifically, we are evaluating the biosynthetic potential of bacteria from ore-forming environments, specifically fluorspar and topaz mines. Soils from ore-forming environments tend have low pH, high saline content, low water holding capacity, and poor nutrient availability. Therefore, ore-forming environments pose a hostile environment for life. To date, no one has explored the natural product potential, or the bacterial diversity, exhibited in these harsh environments. To assess the bacterial diversity, bacteria were isolated from various ore-forming environments using a procedure that is selective for actinobacteria. Following bacterial isolation, genomic DNA was isolated and 16s rRNA gene sequencing was performed to gauge the type of bacteria that were isolated. To stimulate secondary metabolite production, bacteria were then subjected to 7 different media conditions. The supernatant collected from these media conditions were tested against ESKAPE pathogens utilizing the CTSI broth microdilution assay. LC-MS MS analysis was performed for samples exhibiting biological activity. GNPS molecular networking was then utilized to determine potential molecules present in each sample.  Through this process we were able to identify one strain, which we named Streptomyces sp. S1A that exhibited a board range of biological activity (anticancer and antibacterial) and possess a wide array of biosynthetic gene clusters ranging complex macrolides (PKS and NRPS) to terpenes. </p> <p>In summary this multifaced approach to natural product discovery may lead to the discovery of novel antibiotics, enable us to increase production of known or unknown antibiotics through chemical induction, and the characterization of metabolites from Streptomyces sp. S1A will shed insight on the biochemical potential of organisms that inhabit ore-forming environments </p>

Mes travaux de recherche ont eu pour but d’analyser la diversité des microorganismes des trois domaines du vivant présents dans des biofilms phototrophes exposés à l’air, se développant sur des substrats minéraux divers, afin d’essayer, d’une part, de répondre à des questions de diversité et de biogéographie et, d’autre part, d’étudier le processus de colonisation par le biais d’expériences d’exposition contrôlées.J’ai ainsi caractérisé, essentiellement par des approches moléculaires basées sur l'analyse des banques des gènes d'ARNr de la petite sous-unité (SSU rDNAs) et sur des analyses d'empreintes communautaires, la diversité microbienne (procaryote et eucaryote) formant des biofilms matures (exposés depuis plusieurs années) dans plusieurs sites géographiques en Irlande du Nord, en France et en Ukraine, dans la région de Chernobyl. Dans ces biofilms soumis à forte pression sélective, nous avons mis en évidence beaucoup de microorganismes hétérotrophes et phototrophes, mais avec une diversité relativement restreinte en comparaison à d’autres milieux comme les sols ou les systèmes aquatiques. Les archées étaient absentes. Les conditions environnementales auxquelles ce type de biofilm est constamment exposé comme l’irradiation, la dessiccation et la limitation des nutriments sélectionnent des microorganismes qui développent des stratégies pour s’adapter comme, entre autres, la production de pigments. Ce sont des microorganismes fréquemment retrouvés dans des milieux désertiques extrêmes et résistants aussi aux radiations ionisantes qui ont ainsi été identifiés, notamment des Deinococcales et des Actinobacteria, ou encore des champignons ascomycètes (Ascomycota). Parmi les organismes phototrophes, nous avons dénombré des Cyanobacteria, des algues vertes (Chlorophyta) et des Streptophyta. Nous avons mis en évidence que les facteurs environnementaux influencent la composition des biofilms. Toutefois, tandis que la composition de la communauté bactérienne est fortement dépendante de la nature du substrat ou elle se développe, la composition des communautés microbiennes eucaryotes dépend de la distance géographique. Nous avons également mené des expériences de colonisation en exposant un même substrat minéral dans trois sites géographiques en Irlande du Nord et en France. L'analyse de la diversité microbienne lors du processus de colonisation a révélé des changements importants dans la composition des communautés, que ce soit pour les procaryotes ou pour les eucaryotes avec, cependant, des comportements différents de ces deux groupes de microorganismes. Dans le cas des bactéries, on observe une transition des Gammaproteobacteria, qui dominent les temps 0-6 mois et qui correspondent vraisemblablement aux cellules inactives en dispersion, vers des Betaproteobacteria, Bacteroidetes, Alphaproteobacteria et Actinobacteria dans des phases successives de formation du biofilm. Par contre, dès leur détection sur le substrat minéral, les eucaryotes sont massivement dominés par des champignons ascomycètes et basidiomycètes, des algues vertes ainsi que d'autres composantes minoritaires comme des ciliés, étant détectées dans des stades plus tardifs. Nos résultats montrent que les organismes hétérotrophes sont pionniers dans la formation de ces biofilms, ce qui permet d'émettre l'hypothèse qu'ils facilitent l'installation des cyanobactéries et surtout des algues vertes. Ils montrent aussi que le processus d'assemblage des communautés bactériennes dépend du temps de colonisation, alors que le site géographique détermine celui des microorganismes eucaryotes. Ces différences majeures de comportement pourraient être expliquées par des modes de vie différents entre les organismes de ces deux grands groupes. / The major objective of my PhD work was the analysis of the diversity of microorganisms from the three domains of life associated with phototrophic biofilms developing on different mineral substrates exposed outdoors. These studies aimed at answering questions about microbial diversity and biogeography and also at studying the colonization process through controlled exposure experiments. I have thus characterized, essentially by molecular methods based on small subunit (SSU) rRNA gene libraries and fingerprinting analyses the diversity of prokaryote and eukaryote microorganisms forming mature biofilms (exposed for several years) in various geographic sites in Northern Ireland, France and Ukraine, in the Chernobyl area. In these biofilms, subjected to strong selective pressure, we found many heterotrophic and phototrophic microorganisms, but their diversity was limited when compared to that of other environments such as soils or aquatic systems. Archaea were absent from all biofilms. The environmental conditions to which these biofilms are constantly exposed, such as irradiation, desiccation and nutrient limitation select for organisms that develop particular adaptive strategies including, among others, pigment production. The microorganisms identified in these biofilms are also frequently found in extreme, desert environments and are known for their resistance also to ionizing radiation, such as Deinococcales and Actinobacteria or ascomycete fungi (Ascomycota). Among phototrophic lineages, we identified Cyanobacteria, Chlorophyta (green algae) and sometimes Streptophyta. We showed that environmental parameters influenced biofilm microbial communities. However, whereas the bacterial community composition depends on the nature of the substrate, the microbial eukaryotic community composition depends on the geographic distance. We also carried out colonization experiences exposing outdoors the same mineral substrate in three different sites in Northern Ireland and France. The analysis of microbial diversity along the colonization process revealed important changes in community composition both for prokaryotes and eukaryotes, although the behavior of the two groups was different. In the case of bacteria, we observed a transition from Gammaproteobacteria, which dominated the initial 0-6 months and which likely corresponded to inactive dispersive cells, towards Betaproteobacteria, Bacteroidetes, Alphaproteobacteria and Actinobacteria in successive steps of biofilm formation. By contrast, since their detection on mineral substrates, eukaryotes were massively dominated by ascomycete and basidiomycete fungi, green algae and other minor components such as ciliates were detected in later stages of biofilm formation. Our results show that heterotrophic organisms are pioneers in the formation of these biofilms, leading to the hypothesis that they facilitate the settlement of Cyanobacteria and, especially, of green algae. They also show that the process of bacteria community assembly depends on colonization time whereas the geographic site determines that of eukaryotic microorganisms. These major differences might be explained by different lifestyles between organisms of the two groups

Arsenic (As) is a toxic metalloid and the remediation of soils and waters from this contaminant as well as the prevention of future contamination are still pending tasks in Chile. There are bacteria able to live in environments polluted with arsenic, as they have tolerance mechanisms for this metalloid, or even can use it for energy metabolism. The potential tolerance mechanisms include the production of siderophores, metabolites with chelating activity that can decrease the toxicity of metals and metalloids. Although a correlation between siderophore production and metalloid tolerance has been described, the structure of arsenic-binding siderophores and their implications in tolerance have not been elucidated yet. In this work, it is proposed that bacteria isolated from contaminated environments produce arsenic-binding siderophores. The main aims of this work are to study the production of the siderophores by arsenic-tolerant bacteria, to characterize these compounds and to determine their relation with tolerance to arsenic. Fourteen arsenic-tolerant bacteria were isolated from contaminated water, From these, four strains belonging to the species Rhodococcus erythropolis, Arthrobacter oxydans and Kocuria rosea were selected, in addition to the previously isolated Rhodococcus erythropolis S43, for a more detailed study. The isolates were used to produce siderophore extracts, which were then evaluated for their iron- and arsenic-binding activity. To detect the latter, a new method (As-mCAS) was set up, based on the Chrome Azurol S (CAS) test, an assay to detect iron-chelating activity of siderophores. After testing the extracts, R. erythropolis S43 was selected as the strain with the best arsenic-binding activity. For the subsequent chemical characterization, siderophores were produced under control conditions (iron-free M9 medium) and under stress conditions with arsenic (iron-free M9 medium with sodium arsenite). HPLC analysis of the extracts for both culture conditions showed the presence of a single compound with both an iron-chelating and an arsenic-binding activity. Analyses by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) for both culture conditions suggested the main presence of the siderophore heterobactin B. In addition, the genome of strain S43 was sequenced. A cluster of ars-genes was predicted, probably responsible for the arsenic-tolerance of the strain. In addition, a complete gene cluster for heterobactin production was found. However, no significant difference was obtained in the expression of these determinants in the presence or absence of arsenic, suggesting that the production of this siderophore in strain S43 is not responsible for the tolerance to the metalloid.

Os micro-organismos são profícuas fontes de produtos naturais bioativos. Diversos fármacos de importância clínica são de origem microbiana, sendo que a maioria dos antibióticos usados clinicamente é produzida por actinobactérias, principalmente do gênero Streptomyces. A resistência a múltiplas drogas por microorganismos patogênicos e também pelas células tumorais leva à necessidade por novos fármacos antibacterianos e antitumorais. Actinobactérias endofíticas têm demonstrado grande potencial para a busca de produtos naturais bioativos. O presente trabalho relata o estudo químico de duas linhagens de actinobactérias endofíticas, Streptomyces sp. RTd 22 e Streptomyces sp RTd 31, isoladas das raízes de Tithonia diversifolia. As frações ativas nos ensaios biológicos foram fracionadas para a identificação dos compostos bioativos, sendo eles os antibióticos macrolídeos concanamicinas A (S31-1) e B (S31-2), anidro-agliconas das concanamicinas A (S31-3) e B (S31-4), todos produzidos por Streptomyces sp RTd31, e o ionóforo poliéter grisorixina (S22-2), produzido por Streptomyces sp. RTd22. Foi realizado o monitoramento da produção desses compostos bioativos por UPLC-MS através do modo SIM. As concanamicinas A e B tiveram um máximo de produção com 96h, já a grisorixina obteve um máximo com 192h. Outros compostos identificados por desreplicação dos extratos butanólicos de ambas as actinobactérias foram os sideróforos norcardamina (S31-7) e desoxi-nocardamina (S31-8), já o sideróforo desferrioxamina B (S31-9) foi identificado apenas nos extratos butanólicos de Streptomyces sp RTd31. Experimentos de variação do meio de cultivo e co-cultura com bactérias patogênicas foram empregados a fim de estimular a biossíntese de novos compostos, porém nenhum novo metabólito foi identificado. O sequenciamento genético da actinobactéria Streptomyces sp. RTd22 permitiu verificar a presença de vários clusters biossintéticos nesse micro-organismo através da análise feita pelo antiSMASH. Foi possível identificar o cluster da himastatina (S22-4) e da coeliquelina (S22-5), sendo que ambos os compostos não foram biossintetizados nas condições de cultivo utilizadas. O cluster biossintético da grisorixina foi determinado e o experimento de recombinação homóloga para a deleção do gene análogo a flavina mono-oxigenase da nigericina nigC foi realizado. Dois mutantes foram obtidos e um deles foi cultivado para a análise do perfil metabólico por espectrometria de massas. Não houve a produção da grisorixina nem do seu possível precursor pelo mutante, mas outros metabólitos foram produzidos / Microorganisms are prolific sources of bioactive natural products. Several clinically important drugs have microbial origin, and most of the therapeutically used antibiotics are produced by actinobacteria, mainly from the genus Streptomyces. The multidrug resistance observed in pathogenic microorganisms and tumor cells lead to the need for new antibacterial and antitumor drugs . Endophytic actinobacteria have shown great potential in the search for bioactive natural products. This work describes the chemical study of two endophytic actinobacteria strains: Streptomyces sp. RTd 22 and Streptomyces sp RTD 31, isolated from Tithonia diversifolia roots. Active fractions in biological assays were further fractionated for identifying the bioactive compounds, which are: the macrolide antibiotics concanamycins (S31-1) and B (S31-2), anhydrous aglycones of concanamycins A (S31-3) and B (S31-4), all four produced by Streptomyces sp. RTd31, and the ionophore polyether grisorixin (S22-2), produced by Streptomyces sp. RTd22. The production of these bioactive compounds was monitored by UPLC-MS via the SIM mode. Concanamycins A and B had maximum production at 96 h, and grisorixin at 192 h. Other compounds identified by the dereplication of buthanolic extracts of both actinobacteria were the siderophore norcardamine (S31-7) and deoxy-nocardamine (S31-8), the siderophores desferrioxamine B (S31-9) was identified only in buthanolic extracts of Streptomyces sp RTd31. Experiments varying media and co-culture were tested to stimulate the biosynthesis of novel compounds, but nothing new was identified. By genome sequencing of Streptomyces sp RTd22 and antiSMASH analysis it was possible to verify the presence of several biosynthetic clusters in the genome of this strain. It was possible to identify the biosynthetic clusters of himastatin (S22-4) and its analogous compound coelichelin (S22-5); however, these compounds were not biosynthesized in the culture conditions used. The grisorixin biosynthetic cluster was determined, and homologous recombination was performed for deleting the analogue gene of nigericin flavin monooxygenase nigCI. Two mutants were obtained, and one of them was cultured for analyzing its metabolic profile by mass spectrometry. There was no production of grisorixin or its possible precursor by the mutant, but others compounds were produced.

Actinobactérias ocorrem em diversos ambientes e possuem grande potencial na produção de enzimas, antibióticos e fármacos. O presente trabalho teve como objetivo o isolamento, a identificação e a bioprospecção de actinobactérias da rizosfera de milho. Foram isoladas 60 linhagens de actinobactérias de plantas sadias de milho, cultivadas em diferentes regiões do Estado de São Paulo. Os isolados com maior atividade, CCMA30 e CCMA33, foram identificados por meio do seqüenciamento parcial do gene 16S rRNA como pertencentes à espécie Streptomyces griseorubiginosus, nestes isolados foi também detectada a presença de genes PKS.O fracionamento do extrato bruto da linhagem CCMA 33 revelou que a fração 7 (m/z 719-768) com atividade antifúngica, foi submetida a fragmentação (MS/MS), onde pelas buscas no dicionário de produtos naturais confirmou-se a presença de Julichromes Q 6-6, substância sintetizada por policetídeos e de mais duas Julichromes (Q 1-3 e Q 3-3). Estes resultados evidenciam a produção de biocompostos de interesse biotecnológico por actinobactérias da rizosfera. / Actinobacteria occur in different environments and have a great potential in producing enzymes, antibiotics and medical molecules. The present work aimed the isolation, identification and the bioprospection by rhizosphere actinobactérias. Sixty strains were isolated from healthy plants of maize, cultivated in different regions Socorro, Serra Negra and Ribeirão Preto. The two isolates with higher activity CCMA30 and CCMA33 were identified by the partial sequencing of the 16S rDNA gene as belonging to the species Streptomyces griseorubiginosus. In these isolates it was detected the presence of PKS genes. The fractioning of the crude extract of the strain CCMA 33 has revealed that fraction 7 (m/z 719-768) displayed antifungal activity and it was submitted to fragmentation (MS/MS), where by searches in the dictionary of natural products detected the presence of Julichromes Q6-6 and the presence of two other Julichromes (Q1-3 and Q3-3). These results highlight the production of secondary metabolites of biotechnological interest by rhizosphere actinobacteria.

Submitted by Regina Correa (rehecorrea@gmail.com) on 2016-09-21T20:04:08Z No. of bitstreams: 1 DissVG.pdf: 887031 bytes, checksum: 3c091b1ab0f608725eb27c6b2e22b2d5 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-23T18:38:21Z (GMT) No. of bitstreams: 1 DissVG.pdf: 887031 bytes, checksum: 3c091b1ab0f608725eb27c6b2e22b2d5 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-23T18:38:29Z (GMT) No. of bitstreams: 1 DissVG.pdf: 887031 bytes, checksum: 3c091b1ab0f608725eb27c6b2e22b2d5 (MD5) / Made available in DSpace on 2016-09-23T18:38:36Z (GMT). No. of bitstreams: 1 DissVG.pdf: 887031 bytes, checksum: 3c091b1ab0f608725eb27c6b2e22b2d5 (MD5) Previous issue date: 2015-12-14 / Não recebi financiamento / The microbial community plays an essential role in maintaining the ecological balance of soil. Interactions between microorganisms and plants have a major influence on the nutrition and health of the latter, and growth-promoting rhizobacteria can be used to improve plant development through a wide range of mechanisms. Therefore, the objective of the present study was to evaluate bacteria as biological control agents to Phytophthora nicotianae and as growthpromoting agents for citrus rootstocks. Altogether were evaluated 30 bacterial isolates, 11 Bacillus spp., 11 actinobacteria, and 8 lactic acid bacteria. For P. nicotianae control tests at first was performed an bioassay with alfalfa seedling in order to select the most promising bacterial isolates for the biocontrol and, posteriorly, the best isolates were applied in Sunki mandarin (Citrus sunki Hort. ex Tan), and rangpur (Citrus × limonia Osbeck) by seeds and substrate microbiolization methods. It was evaluated the number of surviving plants, at 90 days after sowing and inoculation. The same Isolates also were evaluated in vitro for indoleacetic acid production, phosphate solubilization, and nitrogen (N) fixation. In vivo testing consisted of growth promotion trials of the bacterial isolates that yielded the best results on in vitro tests with three rootstocks: Swingle citrumelo [Citrus × paradisi Macfad cv. Duncan × Poncirus trifoliata (L.) Raf.], Sunki mandarin and rangpur. The parameters of interest were height, number of leaves, stem diameter, shoot and root dry mass, and total dry mass at 150 days after germination. The results showed that alfalfa seedling bioassay vi was able to evaluate the potential for biocontrol of bacterial isolates against to P. nicotianae, BL06 and BL12 (both lactic acid bacteria) showed potential to be used as biocontrol agents of disease, independently from the application method. BM16 and CPMO4 were able to promote growth of Swingle citrumelo. In Sunki mandarin plants, the best treatment results were obtained with BM17 (Bacillus sp.) and ACT11 (actinobacteria). For Rangpur lime rootstock, only BM05 (Bacillus sp.) was able to promote increase in two parameters assessed, height and number of leaves. / A comunidade microbiana tem um papel fundamental na manutenção do equilíbrio ecológico do solo. As interações entre micro-organismos e plantas têm grandes influencias sobre a sanidade e a nutrição das mesmas, nesse contexto, o uso de rizobactérias promotoras de crescimento pode melhorar o desenvolvimento das plantas, por meio de uma ampla variedade de mecanismos. Portanto, o presente trabalho teve como objetivo avaliar bactérias como agentes de controle biológico de Phytophthora nicotianae e como promotoras de crescimento de porta-enxertos de citros. Ao todo foram avaliados 30 isolados bacterianos, sendo 11 Bacillus spp., 11 actinobacterias e 8 bactérias láticas. Para os ensaios de controle de P. nicotianae, incialmente, foram realizados ensaios com brotos de alfafa com a finalidade de selecionar os isolados bacterianos mais promissores para o biocontrole e, posteriormente, os melhores isolados foram aplicados em plantas de tangerina Sunki (Citrus sunki Hort. ex Tan) e limão Cravo (Citrus limonia Osbeck) pelos métodos de microbiolização das sementes e microbiolização do substrato, avaliando-se o número de plantas sobreviventes 90 dias após a semeadura e inoculação. Os 30 isolados bacterianos foram, também, avaliados in vitro quanto à produção de ácido indolacético, solubilização de fosfato e fixação de nitrogênio. Em seguida, para estudo da promoção de crescimento de plantas cítricas, foram realizados testes in vivo utilizando três porta-enxertos, citrumelo Swingle [Citrus paradisi Macfad cv. Duncan x Poncirus trifoliata (L.) Raf.], tangerina Sunki e limão Cravo, sendo utilizados como parâmetros de avaliação, a altura, o número de folhas, o diâmetro do caule, a massa seca da raiz, a massa seca da iv parte aérea e a massa seca total. Pelos resultados obtidos verificou-se que o ensaio realizado com brotos de alfafa permitiu avaliar o potencial de biocontrole das bactérias testadas contra P. nicotianae; os isolados de bactéria lática BL06 e BL12 apresentam potencial para serem utilizados como agentes de biocontrole da doença, independente do método de aplicação utilizado; os isolados de Bacillus spp. BM16 e CPMO4 foram capazes de promover o crescimento do porta-enxerto Citrumelo Swingle, enquanto que, BM17 (Bacillus sp.) e ACT11 (actinobacteria) promoveram o crescimento de plantas de tangerina Sunki. Para o porta-enxerto limão Cravo, apenas BM05 (Bacillus sp.) foi capaz de promover aumento da altura e do número de folhas das plantas.

Actinobactérias ocorrem em diversos ambientes e possuem grande potencial na produção de enzimas, antibióticos e fármacos. O presente trabalho teve como objetivo o isolamento, a identificação e a bioprospecção de actinobactérias da rizosfera de milho. Foram isoladas 60 linhagens de actinobactérias de plantas sadias de milho, cultivadas em diferentes regiões do Estado de São Paulo. Os isolados com maior atividade, CCMA30 e CCMA33, foram identificados por meio do seqüenciamento parcial do gene 16S rRNA como pertencentes à espécie Streptomyces griseorubiginosus, nestes isolados foi também detectada a presença de genes PKS.O fracionamento do extrato bruto da linhagem CCMA 33 revelou que a fração 7 (m/z 719-768) com atividade antifúngica, foi submetida a fragmentação (MS/MS), onde pelas buscas no dicionário de produtos naturais confirmou-se a presença de Julichromes Q 6-6, substância sintetizada por policetídeos e de mais duas Julichromes (Q 1-3 e Q 3-3). Estes resultados evidenciam a produção de biocompostos de interesse biotecnológico por actinobactérias da rizosfera. / Actinobacteria occur in different environments and have a great potential in producing enzymes, antibiotics and medical molecules. The present work aimed the isolation, identification and the bioprospection by rhizosphere actinobactérias. Sixty strains were isolated from healthy plants of maize, cultivated in different regions Socorro, Serra Negra and Ribeirão Preto. The two isolates with higher activity CCMA30 and CCMA33 were identified by the partial sequencing of the 16S rDNA gene as belonging to the species Streptomyces griseorubiginosus. In these isolates it was detected the presence of PKS genes. The fractioning of the crude extract of the strain CCMA 33 has revealed that fraction 7 (m/z 719-768) displayed antifungal activity and it was submitted to fragmentation (MS/MS), where by searches in the dictionary of natural products detected the presence of Julichromes Q6-6 and the presence of two other Julichromes (Q1-3 and Q3-3). These results highlight the production of secondary metabolites of biotechnological interest by rhizosphere actinobacteria.

Os micro-organismos são profícuas fontes de produtos naturais bioativos. Diversos fármacos de importância clínica são de origem microbiana, sendo que a maioria dos antibióticos usados clinicamente é produzida por actinobactérias, principalmente do gênero Streptomyces. A resistência a múltiplas drogas por microorganismos patogênicos e também pelas células tumorais leva à necessidade por novos fármacos antibacterianos e antitumorais. Actinobactérias endofíticas têm demonstrado grande potencial para a busca de produtos naturais bioativos. O presente trabalho relata o estudo químico de duas linhagens de actinobactérias endofíticas, Streptomyces sp. RTd 22 e Streptomyces sp RTd 31, isoladas das raízes de Tithonia diversifolia. As frações ativas nos ensaios biológicos foram fracionadas para a identificação dos compostos bioativos, sendo eles os antibióticos macrolídeos concanamicinas A (S31-1) e B (S31-2), anidro-agliconas das concanamicinas A (S31-3) e B (S31-4), todos produzidos por Streptomyces sp RTd31, e o ionóforo poliéter grisorixina (S22-2), produzido por Streptomyces sp. RTd22. Foi realizado o monitoramento da produção desses compostos bioativos por UPLC-MS através do modo SIM. As concanamicinas A e B tiveram um máximo de produção com 96h, já a grisorixina obteve um máximo com 192h. Outros compostos identificados por desreplicação dos extratos butanólicos de ambas as actinobactérias foram os sideróforos norcardamina (S31-7) e desoxi-nocardamina (S31-8), já o sideróforo desferrioxamina B (S31-9) foi identificado apenas nos extratos butanólicos de Streptomyces sp RTd31. Experimentos de variação do meio de cultivo e co-cultura com bactérias patogênicas foram empregados a fim de estimular a biossíntese de novos compostos, porém nenhum novo metabólito foi identificado. O sequenciamento genético da actinobactéria Streptomyces sp. RTd22 permitiu verificar a presença de vários clusters biossintéticos nesse micro-organismo através da análise feita pelo antiSMASH. Foi possível identificar o cluster da himastatina (S22-4) e da coeliquelina (S22-5), sendo que ambos os compostos não foram biossintetizados nas condições de cultivo utilizadas. O cluster biossintético da grisorixina foi determinado e o experimento de recombinação homóloga para a deleção do gene análogo a flavina mono-oxigenase da nigericina nigC foi realizado. Dois mutantes foram obtidos e um deles foi cultivado para a análise do perfil metabólico por espectrometria de massas. Não houve a produção da grisorixina nem do seu possível precursor pelo mutante, mas outros metabólitos foram produzidos / Microorganisms are prolific sources of bioactive natural products. Several clinically important drugs have microbial origin, and most of the therapeutically used antibiotics are produced by actinobacteria, mainly from the genus Streptomyces. The multidrug resistance observed in pathogenic microorganisms and tumor cells lead to the need for new antibacterial and antitumor drugs . Endophytic actinobacteria have shown great potential in the search for bioactive natural products. This work describes the chemical study of two endophytic actinobacteria strains: Streptomyces sp. RTd 22 and Streptomyces sp RTD 31, isolated from Tithonia diversifolia roots. Active fractions in biological assays were further fractionated for identifying the bioactive compounds, which are: the macrolide antibiotics concanamycins (S31-1) and B (S31-2), anhydrous aglycones of concanamycins A (S31-3) and B (S31-4), all four produced by Streptomyces sp. RTd31, and the ionophore polyether grisorixin (S22-2), produced by Streptomyces sp. RTd22. The production of these bioactive compounds was monitored by UPLC-MS via the SIM mode. Concanamycins A and B had maximum production at 96 h, and grisorixin at 192 h. Other compounds identified by the dereplication of buthanolic extracts of both actinobacteria were the siderophore norcardamine (S31-7) and deoxy-nocardamine (S31-8), the siderophores desferrioxamine B (S31-9) was identified only in buthanolic extracts of Streptomyces sp RTd31. Experiments varying media and co-culture were tested to stimulate the biosynthesis of novel compounds, but nothing new was identified. By genome sequencing of Streptomyces sp RTd22 and antiSMASH analysis it was possible to verify the presence of several biosynthetic clusters in the genome of this strain. It was possible to identify the biosynthetic clusters of himastatin (S22-4) and its analogous compound coelichelin (S22-5); however, these compounds were not biosynthesized in the culture conditions used. The grisorixin biosynthetic cluster was determined, and homologous recombination was performed for deleting the analogue gene of nigericin flavin monooxygenase nigCI. Two mutants were obtained, and one of them was cultured for analyzing its metabolic profile by mass spectrometry. There was no production of grisorixin or its possible precursor by the mutant, but others compounds were produced.