No description available.

The aim of this study was to identify and measure areas of physiology relevant to the initiation of secondary metabolism and spore formation. It was discovered that the growth kinetics of Saccharopolyspora erythraea in a liquid culture environment were dependent on the type of nutrient limitation the organism was exposed to. Sacc. erythraea was able to produce erythromycin and formed structures which had properties similar to those of spores (designated 'sonic resistant units' in this thesis) in a nitrogen limited medium but, would produce erythromycin but no 'sonic resistant units' in a carbon limited medium. This resulted in two liquid culture systems, one which supported the formation of 'sonic resistant units' and one which did not, and thus enabled the comparison of the conditions which resulted in sporulation or secondary metabolism. The measurement of the rate of DNA, RNA and protein synthesis showed distinct profiles of synthesis in both media. Measurements of these rates in Streptomyces hygroscopicus showed similar profiles in the same media suggesting that the behaviour of the macromolecular synthesis rates in different nutrient limiting conditions might be applicable to streptomycetes in general. A change in macromolecular synthesis rate was implicated in inducing secondary metabolism. Antibiotics were used to modulate these rates in an attempt to induce secondary metabolism. Results from these experiments suggested the possible role of the intracellular concentration of charged and uncharged tRNA in induction of antibiotic synthesis. This relationship implies an analogy between the stringent response and the physiology of the induction of secondary metabolism. The formation of spores ('sonic resistant units') did not appear to be induced by changes in macromolecular synthesis rate or changes in tRNA levels or ratio. However, it was evident that the rates of macromolecular synthesis increased during the production of spores and this implied that it is the action of some intracellular agent that is inducing macromolecular synthesis to fuel the spore forming process.

An unknown Saccharopolyspora species which displayed fungicidal activity was selected as a representative of the genus. The antifungal activity could not be repeated in liquid cultures after the initial screen despite several different approaches. A defined medium was developed. However, biomass yields were low (< lg/1) and antifungal activity was not detected. Good growth was observed in nutrient broth (NB) and on nutrient agar (NA). The maximum specific growth rate in NB was 0.25h-1. Growth on NA (measured using image analysis) conformed to the linear growth law with a mean radial growth rate of 0.0327mm/h. Fungicidal activity was measured on solid media, this was presumably due to antibiotic production, although an active compound could not be isolated. A mutation program yielded only non producing mutants despite the mutants having a range of growth rates. A known Saccharopolyspora species, Saccharopolyspora erythraea, was then adopted as a representative of the genus for the remainder of the study. Defined media were developed to permit growth limitation by a range of nutrients. A comparison of several approaches to calculate the maximum specific growth rate was made (ranging from 0.119 to 0.148h-1 in carbon limited media, 0.093 to 0.130h-1 in nitrogen limited, and 0.143 to 0.165h-1 in phosphate limited). Curve fitting provided an accurate series of specific growth rate estimates. The control mechanisms involved in the biosynthesis of erythromycin by Saccharopolyspora erythraea were investigated. By culturing this organism in a range of conditions, and analysing the effects on various growth parameters, nutrient specific patterns of control were observed. Increasing biomass and increasing antibiotic titres occurred concurrently in media when growth was carbon (C), nitrogen (N), and phosphate (P) limited. Generally, when antibiotic titre parallels growth, it is assumed that the product is growth linked. However, further analysis contradicted this. The peaks of specific growth rates and specific erythromycin production rates, obtained by curve fitting, were clearly temporally separated in carbon and phosphate limited conditions indicating non-growth linked production of erythromycin. Growth associated production was confirmed in nitrogen limited conditions. Phosphate limitation supported higher antibiotic yields than the other limitations (Yp/x max mg/g = 6.4 in CL1, 18.5 in NL1, and 28.8 in PL1). Erythromycin production was sensitive to ammonium and to glucose during phosphate limitation. However, this sensitivity to glucose was not obvious in the nitrate limited medium, suggesting different control mechanisms may affect the growth linked and non-growth linked production of erythromycin. Investigations into the regulation of the initiation of erythromycin production indicated that energy charge was not obviously involved, as it appeared to be related to the specific growth rate rather than antibiotic production. The rate of protein synthesis appeared to be a strong candidate for the initiator of antibiotic biosynthesis. Chemostat culture confirmed that erythromycin production was growth linked in nitrate limited conditions. However, the kinetics were more complicated as both biomass and antibiotic decreased with increasing dilution rates. The data conformed to the model proposed by Pirt (1975) for non-competitive inhibition by a growth linked product. A modelling package generated estimates of parameters involved (ki = 1.2mg/1, ks = 0.017g/l, micro max = 0.062h-1 and Yp/s = 0.0019).

No description available.

Psidium guajava é uma espécie tropical, frutífera, de grande importância na fruticultura brasileira e é usada em diversas partes do mundo para o tratamento de doenças que acometem a humanidade. Além de sua importância alimentícia e medicinal, P. guajava também se mostrou boa bioindicadora de poluentes atmosféricos como ozônio e SO2 , pois apresenta respostas características e dose-dependentes das concentrações desses poluentes. Mostrou-se, dessa forma, uma espécie interessante para o estudo de respostas de espécies tropicais a elevadas concentrações de CO2 atmosférico. A fim de avaliar se o elevado CO2 seria uma situação estressante para essa espécie, foi realizada uma fumigação de 130 mudas de P. guajava cv. Pedro Sato em quatro câmaras de topo aberto: duas com ar ambiente ((&sim;390 ppm) e duas com atmosfera enriquecida com CO2 (&sim;720 ppm). Foram realizadas coletas quinzenais, retirando-se cinco indivíduos de cada câmara, aleatoriamente, que foram separados em raiz, caule e folhas e imediatamente congelados com nitrogênio líquido. Foram analisados componentes relacionados a sinalização e inativação de espécies reativas de oxigênio em folhas: os teores de poliaminas solúveis, ácido ascórbico e glutationa, além de fenóis, flavonoides, proantocianidinas e taninos totais. Além disso, os extratos metanólicos de folhas foram submetidos à CLAE para análises quali e quantitativas dos principais flavonoides. Parâmetros relacionados ao desenvolvimento da planta também foram analisados, como incremento em altura, massa seca, fração de massa, acúmulo de carbono, açúcares solúveis e amido. As análises de poliaminas, ácido ascórbico, glutationa, e compostos fenólicos de folhas não revelaram alterações significativas entre os tratamentos, exceto para a análise de taninos totais onde foi observado um aumento significativo dessas substâncias nas plantas mantidas a elevado CO2 e após 90 dias de fumigação. A análise por CLAE revelou que a maioria dos flavonoides dessa espécie é derivada de quercetina. Com relação aos parâmetros de crescimento, P. guajava não apresentou diferenças significativas de incremento em altura e massa seca, mas apresentaram acúmulo de amido nas folhas. Analises multivariadas foram utilizadas para integrar as 46 variáveis analisadas. Neste estudo viu-se que a situação de elevado CO2 em mudas de P. guajava é favorável, visto que ocorre acúmulo de amido, substâncias anti-herbivóricas (taninos) e, aparentemente, não há alteração no balanço redox. Contudo, é de extrema importância estudos que avaliem alterações concomitantes de parâmetros ambientais, como aumento de temperatura, disponibilidade de água, ozônio e elevado CO2 . A goiabeira apresenta grande importância econômica relacionada à produção e qualidade de seus frutos, dessa forma são necessários estudos que avaliem o efeito do aumento do CO2 na quantidade e qualidade de frutos produzidos por esta espécie. Por ser uma espécie de crescimento lento, períodos de exposição mais longos são aconselháveis para uma melhor analise da susceptibilidade dessa espécie ao elevado CO2 / Psidium guajava is a tropical fruit species of great importance in Brazilian economy; it is also used in several parts of the world as medicinal. Besides its importance as medicinal and nutritional species, P. guajava has also showed to be a good bioindicator of air pollutants, such as ozone and SO2 . It presents characteristic responses, dose-dependent of the concentrations of these pollutants. Therefore, this species seems to be interesting to study the responses of a tropical species to high concentrations of atmospheric CO2 . In order to assess whether the high CO2 would be a stressful situation for this species, we performed a fumigation experiment using 130 seedlings of P. guajava cv. Pedro Sato in four open-top chambers: two with ambient air (&sim; 390 ppm CO2 ) and two with elevated CO2 (&sim; 720 ppm). Biweekly, five individuals of each chamber were randomly collected, separated into root, stem and leaves and immediately frozen with liquid nitrogen. Were analyzed parameters related to signaling and inactivation of reactive oxygen species in leaves: levels of soluble polyamines, ascorbate and glutathione, as well as contents of phenols, flavonoids, tannins and proanthocyanidins. Furthermore, the methanol extracts were subjected to HPLC for quantitative and qualitative analysis of the major flavonoids. Parameters related to the development of the plant were also analyzed, as height increment, dry mass, mass fraction, carbon accumulation, soluble sugars and starch accumulation. The analysis of polyamines, ascorbate, glutathione and phenolic compounds of leaves, showed no significant changes when comparing treatments, except for the analysis of tannins which was observed a significant increase of these substances in plants at high CO2 after 90 days of fumigation. HPLC analysis revealed that most of flavonoid composition is quercetin derivatives. The growth parameters showed no significant differences in height increment and dry mass, but the leaves showed accumulation of starch after CO2 fumigation. Multivariate analyzes were used to integrate the 46 variables analyzed. In this study it was seen that the elevated CO2 seem to be favorable to seedlings of P. guajava, once there is an accumulation of starch, antiherbivore substances (tannins) and, apparently, there was no change in the redox balance. However, it is extremely important to perform studies that evaluate concomitant changes in environmental conditions, such as increased temperature, availability of water, ozone and elevated CO2 . Guava has great economic importance in Brazil, fact related to the production and quality of its fruits, so more studies are needed to assess the effect of increased CO2 in the quantity and quality of fruits produced by this species. As a slow-growing species, longer exposure periods are recommended to better analyze the susceptibility of this species to elevated CO2

Bacterial secondary metabolite biosynthetic pathways are frequently encoded in gene clusters. Genomic sequence information allows the identification of likely biosynthetic clusters based on sequence homology to known proteins. Biochemical characterization of suspected biosynthetic enzymes affords the discovery of pathways which may never be identified by traditional screening approaches. In the work presented here, I, in some cases in collaboration with others, characterize the three intragenomic GTP cyclohydrolase II (GCH II) homologs from Streptomyces coelicolor A3(2) and show that one catalyzes a related but distinct reaction from the other two. The basis for the altered activity is investigated and speaks to the chemical mechanism of not only the unusual enzyme but also to all GCH II enzymes. Further, I investigate two other enzymes found in the same gene cluster as the unusual GCH II. Using biochemical techniques, I show that the product of the unusual GCH II is used as a substrate by a creatinine amidohydrolase homolog. Using structural biology, I show that the third enzyme, a 6-pyruvoyltetrahydropterin synthase (PTPS), can not catalyze the PTPS reaction but is capable of binding a pterin substrate. Finally, I propose that the cluster from S. coelicolor containing the unusual GCH II encodes enzymes for a novel pathway to produce a pterin.

Made available in DSpace on 2014-06-11T19:32:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-11Bitstream added on 2014-06-13T19:03:08Z : No. of bitstreams: 1 ehlert_pad_dr_botfca.pdf: 437229 bytes, checksum: f0831a89a35fb6fe8519d87cf7342229 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este trabalho foi elaborado visando determinar a influência da idade e horário de colheita para a erva-cidreira brasileira, Lippia alba (Mill.) N. E. Br., quimiotipo carvona-limoneno, sobre a produção de massa foliar, rendimento e composição do óleo essencial, bem como os teores e quantidades extraídas dos macronutrientes. O trabalho constou de dois experimentos, no primeiro testou-se diferentes idades de colheita em quatro épocas de plantio, e no segundo cinco horários de colheita, porém somente em uma época. Os experimentos foram estabelecidos na Fazenda Experimental Lageado da FCA-UNESP/Botucatu no Setor de Horticultura do Departamento de Produção Vegetal. Os plantios do primeiro ensaio foram realizados nas seguintes épocas: outubro de 2000, e janeiro, maio e julho de 2001, constando de nove idades de colheita por época. O delineamento experimental utilizado foi o de blocos casualizados, através de um arranjo fatorial 9x4, sendo nove idades de colheita em quatro épocas de plantio com quatro repetições cada. Cada parcela tinha 30 plantas, sendo 12 plantas úteis e um total de 888 plantas para cada época. A colheita foi realizada a cada 15 dias, a partir dos 100 dias após o plantio até completar 220 dias. Neste ensaio também se verificou os teores e a extração dos macronutrientes das folhas. O segundo ensaio constou de cinco horários: às 8:00, 10:00, 12:00, 14:00 e 16:00 h com cinco repetições, distribuídos em blocos casualizados, sendo efetuada somente uma colheita aos 145 dias. As colheitas para ambos experimentos foram realizadas a 15 cm da superfície do solo... . / This work was elaborated with the purpose to determine the influence of age and schedules of harvest for the Brazilian lemon verbena, Lippia alba (Mill.) N.E. Br., chemotype carvone-limonene, its foliar mass production, efficiency and composition of the essential oil, as well as, the contents and quantities extracted form the macronutrients through their leaves. The work consists of two experiments, in the first one, different intervals of crops were tested in the four seasons of the year, in the second one, five different schedules of cropping, but, only in summer. The experiments were established at the Experimental Farm of Lageado of FCA-UNESP/Botucatu in the Horticulture Sector of the Department of Vegetable Production. The plantings of the first testing were done in the following seasons: October 2000, January, May and July 2001, being observed 9 harvest ages each season. The experimental delineation used was of chance blocks, through a 9X4 factor arrangement, being 9 crop intervals in 4 seasons with 4 repetitions of each. Each portion had 30 plants, being that 12 plants were useful and a total of 888 plants for each season. The harvest was accomplished every 15 days, starting after 100 days of age, until it completed 220 days. In this trial the tenor and the extraction of the macronutrients of the leaves were also verified. The second trial was of the time of the.harvest which had five schedules: at 8:00, 10:00, 12:00, 14:00 and 16:00 hrs with 5 repetitions, distributed in 5 chance blocks, only one harvest being done at 145 days. The harvests for both experiments were done at 15 cm from the soil surface... (Complete abstract, click electronic address below).

Resumo: Este trabalho foi elaborado visando determinar a influência da idade e horário de colheita para a erva-cidreira brasileira, Lippia alba (Mill.) N. E. Br., quimiotipo carvona-limoneno, sobre a produção de massa foliar, rendimento e composição do óleo essencial, bem como os teores e quantidades extraídas dos macronutrientes. O trabalho constou de dois experimentos, no primeiro testou-se diferentes idades de colheita em quatro épocas de plantio, e no segundo cinco horários de colheita, porém somente em uma época. Os experimentos foram estabelecidos na Fazenda Experimental Lageado da FCA-UNESP/Botucatu no Setor de Horticultura do Departamento de Produção Vegetal. Os plantios do primeiro ensaio foram realizados nas seguintes épocas: outubro de 2000, e janeiro, maio e julho de 2001, constando de nove idades de colheita por época. O delineamento experimental utilizado foi o de blocos casualizados, através de um arranjo fatorial 9x4, sendo nove idades de colheita em quatro épocas de plantio com quatro repetições cada. Cada parcela tinha 30 plantas, sendo 12 plantas úteis e um total de 888 plantas para cada época. A colheita foi realizada a cada 15 dias, a partir dos 100 dias após o plantio até completar 220 dias. Neste ensaio também se verificou os teores e a extração dos macronutrientes das folhas. O segundo ensaio constou de cinco horários: às 8:00, 10:00, 12:00, 14:00 e 16:00 h com cinco repetições, distribuídos em blocos casualizados, sendo efetuada somente uma colheita aos 145 dias. As colheitas para ambos experimentos foram realizadas a 15 cm da superfície do solo... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: This work was elaborated with the purpose to determine the influence of age and schedules of harvest for the Brazilian lemon verbena, Lippia alba (Mill.) N.E. Br., chemotype carvone-limonene, its foliar mass production, efficiency and composition of the essential oil, as well as, the contents and quantities extracted form the macronutrients through their leaves. The work consists of two experiments, in the first one, different intervals of crops were tested in the four seasons of the year, in the second one, five different schedules of cropping, but, only in summer. The experiments were established at the Experimental Farm of Lageado of FCA-UNESP/Botucatu in the Horticulture Sector of the Department of Vegetable Production. The plantings of the first testing were done in the following seasons: October 2000, January, May and July 2001, being observed 9 harvest ages each season. The experimental delineation used was of chance blocks, through a 9X4 factor arrangement, being 9 crop intervals in 4 seasons with 4 repetitions of each. Each portion had 30 plants, being that 12 plants were useful and a total of 888 plants for each season. The harvest was accomplished every 15 days, starting after 100 days of age, until it completed 220 days. In this trial the tenor and the extraction of the macronutrients of the leaves were also verified. The second trial was of the time of the.harvest which had five schedules: at 8:00, 10:00, 12:00, 14:00 and 16:00 hrs with 5 repetitions, distributed in 5 chance blocks, only one harvest being done at 145 days. The harvests for both experiments were done at 15 cm from the soil surface... (Complete abstract, click electronic address below). / Orientador: Lin Chau Ming / Coorientador: Dirceu Maximino Fernandes / Doutor

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique. IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Actinobacteria, primarily the genus Streptomyces, have led to the development of a number of antibiotics, which result from their secondary metabolites or modified derivatives. Secondary metabolite production can result from competition with neighboring microbes in an effort to disrupt growth, aiding in the competition for vital nutrients in impoverished conditions. Such secondary metabolites have the potential to affect a plethora of cellular functions in target cells, including, cell wall development, protein synthesis, protein function and fatty acid synthesis/metabolism. Due to the pandemic spread of antibiotic resistant bacteria, it is imperative to continue the search for new therapeutic agents targeting these deadly organisms. As such, our group explored soil and marine samples from Tampa Bay’s surrounding farmlands and waterways for secondary metabolite producing microbes using culture methods specific to Actinobacteria. Through these efforts we isolated over 750 bacterial species, of which almost half are confirmed Actinobacteria. In an attempt to derive new and novel chemistry from these organisms, we used our novel collection, and developed techniques for epigenetic modification to un-silence dormant and cryptic metabolic pathways. Our work reveals that a number of these Actinobacteria produce secondary metabolites that are effective against the ESKAPE pathogens, some at very low concentrations. Although the bioactivity from secondary metabolites is a well-known source for antibiotic drug discovery, our epigenetic methods suggest a potential to isolate previously overlooked compounds that have a very real possibility for use as antibacterial therapeutics.