Köln, University, Diss., 2001.

Mainz, Universiẗat, Diss., 2001.

Bochum, Universiẗat, Diss., 2002.

Düsseldorf, Universiẗat, Diss., 2002.

München, Techn. Universiẗat, Diss., 2002.

Braunschweig, Techn. Universiẗat, Diss., 2002.

Kaiserslautern, Techn. Universiẗat, Diss., 2004.

Orientador: Wanderley Dias da Silveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-25T14:11:51Z (GMT). No. of bitstreams: 1 Benetti_Fabiane_M.pdf: 10091748 bytes, checksum: 69f74749fc8856af3a4d0c1b08a3f49b (MD5) Previous issue date: 1999 / Resumo: Treze linhagens de Escherichia coZi, isoladas de pacientes do Hospital das Clí nicas da Universidade Estadual de Campinas (UNICAMP) que apresentavam infecções do trato urinário, foram estudadas com relação às suas características bioquímica (perfil eletroforético de isoenzimas), moleculares (perfil plasmidial, perfil de DNA segundo técnica de ribotipagem-RFLP e mutagenicidade) e de patogenicidade (produção de hemolisina, produção de aerobactina, absorção do corante vermelho de Congo e capacidade de adesão e invasão in vitro em células HeLa). Uma linhagem foi mutagenizada com a finalidade de localizar possíveis genes envolvidos com patogenicidade. Os dados obtidos com os perfis eletroforéticos de isoenzimas e ribotipagem permitiram a construção de dendogramas de dessemelhanças das diferentes linagens. Com exceção à capacidade de adesão nas linhagens celulares cultivadas in vitro, nenhuma das demais características pode ser relacionada à patogenicidade / Abstrat: Thirteen E. coZi strains isolated from Clínical Hospital patients of the State University of Campinas (UNICAMP) that suffered of urinary tract infection or pyelonephritis, were studied regarding their biochemical characterisitic (isoenzymes eletrophoretic profile), molecular characteristics (plasmid profiles, DNA profile by PFLP of rRNA) and pathogenicity characteristics (hemolysin and aerobactin production, in vivo absorption of Congo red dye and in vivo capacity of adherence and invasion in HeLa cells). One strain was mutagenised in order of locating possible genes involved with pathogenicity. The results obtained by isoenzymes and RFLP eletrophoretics profiles allowed the construction of dissimilarity dendograms os differents strains. With the exception of adherence capacity, none of the others studied characteristics could be associated with pathogenicity / Mestrado / Microbiologia e Imunologia / Mestre em Ciências Biológicas

Memoria para optar al título profesional de Bioquímico / Diversas investigaciones han mostrado que cultivos microbianos provenientes de una cepa pura pueden dar paso gradualmente a la emergencia y mantención de diversidad genética en condiciones de privación de recursos. En fase estacionaria de largo plazo, en Escherichia coli, se ha observado que aquellas mutantes con mayor adecuación biológica son capaces de invadir el sistema (selección periódica), lo cual posteriormente da paso a una fase de coexistencia y como consecuencia la biodiversidad se ve incrementada. En este trabajo se postula que el mecanismo responsable de ambas dinámicas, selección periódica y coexistencia, es el cross-feeding, en otras palabras la existencia de acoplamientos entre los requerimientos de nutrientes por parte de las mutantes, tal que los productos de excreción de un genotipo son los recursos de otro. El objetivo de esta tesis fue estudiar el rol de este mecanismo en las dinámicas de diversidad observadas en fase estacionaria de largo plazo, a través de la generación de un modelo de simulación matemático. En el modelo propuesto, las mutaciones le confieren a los individuos la capacidad de metabolizar y excretar distintos metabolitos, donde la supervivencia y la reproducción son dependientes de los recursos requeridos por cada genotipo. Nuestros resultados muestran que bajo condiciones limitadas de nutrientes, en ausencia de cross-feeding, la población muere rápidamente. Sin embargo, la presencia de cross-feeding produjo dinámicas de diversidad similares a las descritas en cultivos de largo plazo (selección periódica y coexistencia). Estos resultados indican que este mecanismo podría ser fundamental en la persistencia de las poblaciones por largos periodos de tiempo y que jugaría un rol importante en la emergencia y mantención de diversidad en ecosistemas microbianos. / Several studies have shown that microbial cultures from a pure strain may gradually lead to the emergence and maintenance of genetic diversity in deprivation of resources. Long-term stationary phase cultures of Escherichia coli have shown that these mutants with higher fitness are able to invade the system (periodic selection), but then these mutants do not exclude each other and as a result biodiversity increases. This work argues that the mechanism responsible for both dynamics, periodic selection and coexistence, is cross-feeding; in other words the existence of links between nutrient requirements by the mutants, in such a way that excretory products of one genotype are the resources of another. The aim of this thesis was to study the role of this mechanism in diversity dynamics observed in long-term stationary phase, through the generation of a mathematical simulation model. In this model, mutations grant individuals the ability to metabolize and excrete different metabolites, where survival and reproduction are dependent on the resources required by each genotype. Our results show that under limited concentrations of nutrients and without recycling of metabolites, the population quickly dies. However, the presence of cross-feeding produced diversity dynamics similar to those observed in long-term batch cultures (periodic selection and coexistence). These results indicate that the proposed mechanism could be fundamental to the persistence of populations for long periods of time and would play an important role in the emergence and maintenance of diversity in microbial ecosystems

Biosynthetic reactions in micro-organisms normally are under precise control, usually by a feed-back mechanism in which the end product inhibits the activity and/or represses the formation of the enzyme which initiates the biosynthetic pathway. Streptomycin (Sm)-dependent mutants of Escherichia coli, unlike the parent wild-type strains excrete the amino acid, L-valine, thereby indicating a loss of precise regulation in the biosynthesis of this amino acid (Tirunarayanan, Vischer and Renner, 1962; Bragg and Polglase, 1962). The initiating enzyme for valine biosynthesis, acetohydroxy acid (AHA) synthetase, is derepressed in Sm-dependent E. coli (Coukell and Polglase, 1965), thus providing an explanation for the excretion of valine by this mutant. However, it was apparent that the extent and cause of regulatory insufficiency in Sm-dependent E. coli required further investigation. A study of the effect of the carbon source used for growth on AHA synthetase formation revealed that in wild-type E. coli B this enzyme was subject to catabolite repression. End-product inhibition of AHA synthetase by L-valine in E. coli B attained a maximum at 60-70% inhibition. These previously unreported properties of AHA synthetase (sensitivity to catabolite repression and incomplete end-product inhibition) are significant in the regulation of the biosynthetic pathway leading to the aliphatic amino acids and pantothenate. In Sm-dependent E. coli B, growing with non-limiting antibiotic, catabolite repression of AHA synthetase was relaxed. Additional evidence for relaxation of catabolite repression in Sm-dependent E. coli was provided by the observation that Sm-dependent mutants of E. coli (strains B and E) were inducible for β-galactosidase in the presence of glucose Furthermore, several glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and iso-citrate dehydrogenase) were found to be insensitive to variation in the nature of the carbon source in a Sm-dependent mutant. Cell yield experiments revealed that aerobic glucose metabolism in the Sm-dependent mutant was one-third less efficient than in the Sm-sensitive strain, although the two strains were equally efficient under anaerobic conditions. Moreover, the rate of ATP synthesis in the Sm-dependent mutant was less than that of the wild-type parent organism. Therefore, relaxation of catabolite repression in Sm-dependent E. coli B appears to result from an impairment of aerobic energy metabolism in this mutant. Catabolite repression in Sm-dependent E. coli B under conditions of antibiotic-limitation was investigated. The growth rate of Sm-dependent E. coli B on limiting concentrations of dihydrostreptomycin (DHSm) was evaluated by means of a constant (K DHSm ) relating half-maximal growth rate to antibiotic concentration. K DHSm varied with the nature of the carbon source being highest with energy-rich compounds (e.g. gluconate) and lowest with energy-poor compounds (e.g. lactate). Glucose-sensitive enzymes of Sm- dependent E. coli B were specifically repressed by antibiotic-limitation and exhibited specific activities lower than those observed for the same enzymes in glucose-grown extracts of wild-type E. coli B. Parallelism was observed between decreasing antibiotic concentration, decreasing growth rate, and increasing catabolite repression of certain glucose-sensitive enzymes (notably AHA synthetase and fumarase). The decreased efficiency of aerobic glucose metabolism of Sm-dependent E. coli B was not affected by variation in the concentration of antibiotic. Thus, it is improbable that carbohydrate metabolism is the antibiotic dependent site in the Sm-dependent mutant. The results are compatible with the hypothesis of Spotts and Stanier (1961) that the primary site of action of DHSm in the Sm-dependent organism is the ribosome (i.e . , protein synthesis). However, the growth-limiting effect of antibiotic deprival appears to be augmented by catabolite repression. Additionally, the Sm-dependent mutant is deficient in energy metabolism which can explain the relaxation of control by catabolite repression when antibiotic is present in non-limiting concentration. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate