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

The succinate oxidase system of E. coli has been studied from three main viewpoints: (a) the preparation and properties of succinate dehydrogenase (SDH), (b) the function of nonheme iron, and (c) the sequence of the components of the respiratory chain. Three different preparations containing SDH activity were isolated from this organism. These were the particulate fraction, soluble respiratory complex, and soluble SDH. The partial purification and characterization of these enzymes or complexes was performed. The particulate fraction consisted of membrane fragments which contained the whole respiratory chain and which oxidized succinate and NADH. The soluble respiratory complex contained both SDH and cytochrome b(1). The molecular weight was 1.6 x 10(6). The soluble SDH did not contain cytochrome b(1) and had a molecular weight of 100,000. One of the characteristic properties of SDH of the particulate fraction and the soluble respiratory complex was activation. If the enzyme was prepared in phosphate buffer both succinate oxidase and SDH activities could be activated by heating at 38° in the presence of succinate. The enzyme was stabilized by succinate in the absence of heating. Activation of succinate oxidase seemed to be mainly due to the activation of SDH. A second activation phenomenon which was independent of heat treatment was also observed. When the enzyme was prepared in Tris buffer with succinate the activated enzyme was formed at 0°. Heating did not further increase its activity. Activation by heat was irreversible. The heat-activated enzyme deactivated to a form which could not be reactivated. The heat-independent activated enzyme was more stable. The two activation phenomena thus seemed to be different. In contrast, the soluble SDH did not show the activation phenomenon nor was it stabilized by substrate. A mechanism for the activation of SDH is proposed. The nature, properties, role and location of nonheme iron in the particulate fraction of E. coli was investigated. The level of nonheme ferrous or ferric iron in the particulate fraction was determined spectrophotometrically using o-phenanthroline or Tiron. Analysis of iron by both chemical and spectrophotometric methods showed that only 45% of the total iron reacted with o-phenanthroline ("o-phenanthroline-reacting iron"). Heme iron constituted 5% of the total iron. The rest of total iron was not exposed by treating the particulate fraction with detergents or urea. The nature of the remainder of the total iron (50%) is unknown. Half of the o-phenanthroline-reacting iron reacted directly with o-phenanthroline ("directly-reacting iron"), but the other half only reacted after addition of dithionite ("dithionite-reducible iron"). Directly-reacting iron appeared to be ferric iron which was located in the hydrophobic region of the particulate fraction. This ferric iron could be reduced by sulfhydryl groups of the protein, The dithionite-reducible iron was probably located at the surface of the particulate fraction and could not be reduced by sulfhydryl groups. Part of the dithionite-reducible iron was reduced by NADH or succinate. This substrate-reducible iron, probably less than 10% of the total iron, was located in the cytochrome b(1) region of the respiratory chain. It was not associated with SDH. The effect of ultraviolet irradiation, inhibitors and extraction of ubiquinone on the activities of SDH and succinate oxidase was examined. From these experiments, and those outlined above, a scheme for the sequence of the succinate oxidase chain of E. coli is proposed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Nucleotide pools in a synchronized culture of Escherichia coli B/r/l oscillate as a function of age. Purine nucleotides showed a gradual increase from zero age to the time of subsequent division, with a maximal 50% increase immediately prior to division. In contrast, pyrimidine nucleotides underwent a diamatic increase of about 50% in the first half of the generation cycle, declining at a time coincident with the termination of a round of DNA replication. A second 50 - 70% increase started at the time of the onset of DNA replication and continued towards cell division, as did the purine. The fluctuation of pyrimidines between zero age and the middle of the division cycle suggests a functional relationship between pyrimidine pool fluctuations and the regulation of DNA replication. Nucleotide pools decrease immediately in the presence of chloramphenicol to 10% of the control concentrations, and overshoot 50 - 70% in restoration of protein synthesis. Feedback inhibition of carbomyl phosphate synthesis (which is required for pyrimidine biosynthesis) by excess arginine may explain the fluctuations of nucleotide pools in the presence of chloramphenicol. Immediate depletion of nucleotide pools could be du to a very rapid turnover of nucleotide biosynthetic enzymes. The depletion of precursor pools, may explain the inability of a cell to reinitiate DNA replication in the absence of protein synthesis. In a comparison experiment, however, nucleotide pools in a temperature-sensitive initiator mutant were seen to accumulate at non-permissive temperature. In this case, protein synthesis occurred but initiation of DNA synthesis could not take place. This confirms the current hypothesis that a functional initiator protein is required for reinitation. Nucleotide pools in the presence of nalidixic acid dropped slightly and although no DNA synthesis occurred, pools showed no accumulation. This suggested a secondary effect of the inhibitor. Experiments prior to the pool analyses showed the importance of balanced growth in such studies. The cell size distribution was seen to be a more valid criterion than exponential increase in numbers. Exposure to cold temperatures was seen to upset balanced growth for at least one generation. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The capsular polysaccharides of Escherichia coli are immunogenic and antigenic. When conjugated to a carrier protein these polysaccharides can be used as vaccines. A knowledge of the structure of bacterial capsular polysaccharides is essential for understanding antibody-antigen interaction and also for understanding the chemical basis of serological differentiation. For these reasons structural studies of some E. coli capsular polysaccharides are being undertaken in this laboratory. In this thesis a preliminary investigation into the structures of capsular polysaccharides from E. coli serotypes K/46, K/47, K/48, K49 and K50 is presented. The qualitative composition of each polysaccharide was determined by varying the hydrolytic conditions used to cleave the glycosidic bonds between the monosaccharide units. Table I shows the sugars present in each capsular polysaccharide. [Table Omitted] The ratio of the sugars in each polysaccharide was determined using methanolysis followed by reduction with sodium borohydride. The presence of amino sugars was confirmed by deamination of the hydrolyzed polysaccharide and detection of the product by g.l.c. G.l.c.-m.s., of the alditol acetates of the monosaccharides obtained by hydrolysis of the polysaccharides, was used to confirm the type of monsaccharide. present in each polysaccharide, ₁H-N.m.r. spectroscopy was utilized to confirm the presence of deoxy sugars, amino sugars and non-carbohydrate substituents. E. coli K47 and K50 capsular polysaccharides were both found to have pyruvate present as a substituent. A bacteriophage was isolated from sewage for each of E. coli K47, K48 and K49 serotypes. Phage 47 also attacked E. coli K48 and K49 bacteria. The structure of E. coli K26 capsular polysaccharide was investigated using the techniques of acid hydrolysis, carbodiimide reduction and methanolysis followed by reduction with sodium borohydride. The polysaccharide was degraded using a bacteriophage-borne glycanase. The position of cleavage found by methylation of the reduced oligosaccharide. Combination of the data obtained from the chemical analysis, n.m.r spectroscopy and the bacteriophage degradation gave the two following possible structures for the E. coli K26 capsular polysaccharide. [Formula Omitted] / Science, Faculty of / Chemistry, Department of / Graduate

Diseases caused by encapsulated bacteria such as E. coli are among the most prevalent in the world. The polysaccharide capsule (K antigen) is an important factor in the virulence and pathogenicity of E. coli bacteria. Serological classification of these bacteria is also based mainly on the immunologically dominant capsular polysaccharide, due to its location at the bacterial cell surface. In order to understand the role played by the K antigens in bacterial infections, and the chemical basis of serological differentiation, the systematic structural investigation of all the capsular polysaccharides of E. coli (74 serotypes) is underway in this laboratory and others. Presented in this thesis are the structures of the K antigens of E. coli K26 and K49 serotypes. K26 capsular polysaccharide was studied using techniques such as methylation analysis, β-elimination, Smith degradation and partial hydrolysis. The oligosaccharides produced by the partial acid hydrolysis were analysed by g.c.-c.i.-m.s. To aid in the characterization of these oligosaccharides, a 'library' of relative retention times and c.i. mass spectra of authentic standards (di-, tri-, and tetra-saccharides) was prepared. The results from these analyses, together with data from n.m.r. spectroscopy of the native polysaccharide and derived oligosaccharides, allowed the following structure to be assigned to E. coli K26 polysaccharide. [Formula Omitted] E. coli K49 capsular antigen contains two amino acids, serine and threonine, amidically linked to the carboxyl group of glucuronic acid. Techniques used in the structural elucidation were raethylation analysis, acetolysis, amino acid analysis, HF hydrolysis, partial acid hydrolysis and Smith degradation. The oligosaccharides generated by the three latter methods were analysed by g.c.-c.i.-m.s. and n.m.r. spectroscopy. A bacteriophage-associated enzyme degradation of the K49 antigen yielded a product (P1) which consisted of a single repeating unit (see below). Results from the analyses of P1 and the chemically produced oligosaccharides were in agreement with the following assignment for the structure of the E. coli K49 capsular polysaccharide. [Formula Omitted] / Science, Faculty of / Chemistry, Department of / Graduate

Orientação: Wanderley Dias da Silveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-20T02:07:58Z (GMT). No. of bitstreams: 1 Manfio_GilsonPaulo_M.pdf: 8642301 bytes, checksum: b93aa6979c378ac694a0d6d30bca8b05 (MD5) Previous issue date: 1990 / Resumo: Amostras de Escherichia coli portadoras das fímbrias FY e 31A, associadas a diarréia e septicemia em bovinos, foram estudadas visando determinar a localização da expressão gênica destas fímbrias. As amostras bacterianas 31A+: BZ43, BZ2468 e 31A e FY+: Att25, 54-5, 2147-4 e 11a, foram analisadas quanto à produção de enterotoxinas (STI e LT), VT, hemolisina, resistência a antibióticos, hemaglutinação D-manose resistente com eritrócitos e reação com antissoros específicos. A caracterização das fímbrias envolveu microscopia eletrônica, análise eletroforética de proteínas totais, de superfície e de membrana em SDS-PAGE e "Western Blotting". Foi também verificada a presença de plasmídios conjugativos nas amostras bacterianas e a caracterização dos transconjugantes para a expressão de FY e 31A. Uma amostra portadora de fímbria 31A (amostra BZ43) foi mutagenizada com transposon TnphoA. A expressão de 31A foi avaliada através de aglutinação com antissoros específicos, hemaglutinação de eritrócitos de boi, adesão a células Hela em cultura de tecido e patogenicidade em teste de competição em camundongo. A análise dos padrões de hemaglutinação, proteínas totais, proteínas de superfície e aglutinação com antissoros específicos permitiu a determinação das subunidades protéicas destas fímbrias como sendo aproximadamente 20 kDa para FY e 17 kDa para 31A. A amostra 11a, anteriormente classificada como portadora de fímbria FY, foi reclassificada como portadora de fímbria 31A através dos resultados de SDS-PAGE de proteínas de superfície e reação com antissoros específicos. As amostras Att25, BZ2468 e 31A apresentaram plasmídios conjugativos com marcas de resistência a antibióticos, porém não codificaram para a expressão de fímbrias nas linhagens transconjugantes, sugerindo que a expressão destas fímbrias ocorre a nível cromossômico. A amostra BZ43 mutagenizada com TnphoA originou duas amostras não-hemaglutinantes (eritrócitos bovinos) que apresentaram reação com o anticorpo específico anti-31A. A perda da capacidade de hemaglutinação foi relacionada à ausência da subunidade da fímbria 31A (17 kDa) em SDS-PAGE de proteínas de superfície. Uma das amostras mutantes apresentou fímbrias em microscopia eletrônica. A ausência defímbrias no outro mutante foi relacionada à não expressão de uma proteína de membrana de 35 kDa. A perda da capacidade de eritrócitos de bovino, associada ou não aglutinação de à expressão de fímbrias, não alterou o padrão de adesão a células HeLa dos mutantes (AD). A amostra BZ43 e o mutante fimbriado não apresentaram patogenicidade no teste de competição em camundongos de 3 dias de idade. A perda da capacidade de hemaglutinação parece não conferir uma menor adesão da amostra mutante nestes animais / Abstract: Escherichia coli strains associated with diarrhea and septicemic infections of cattle were studied to determine the genetic expression of FY and 31A fimbriae. The strains 31A+: BZ43, BZ2468 and 31A, and FY+: Att25, 54-5, 2147-4 and 11a, were evaluated for enterotoxin production (STI and LT), VT, hemolysin, antibiotic resistance, mannose resistant RBC hemagglutination, and reaction with specific antisera. The characterization of fimbriae involved electron microscopy, SDS-PAGE of total, surface and membrane proteins and Western blotting. Conjugative plasmids in the strains were transferred and analyzed for FY and 31A expression. Hemagglutination, SDS-PAGE of total and surface proteins and reaction with specific antisera allowed the location of the fimbrial subunits at 20 kDa for FY and 17 kDa for 31A. Strain 11a, originally grouped as FY by others, was classified as 31A based on SDS-PAGE of surface proteins and reaction with specific antisera. Strains Att25, BZ2468 and 31A had conjugative plasmids coding antibiotic resistance marks, but transconjugants failed to express the fimbriae, suggesting it could be located in the bacterial chromosome. Strain BZ43 (31A) was mutagenized using TnphoA transposon and mutants with negative hemagglutination of bovine RBC were assayed with specific antiserum, and tested for adhesion to HeLa cells in tissue culture and pathogenicity in the mouse competition assay. Loss of hemagglutinating capacity was related to the lack of the 31A fimbriae subunit (17 kDa) on SDS-PAGE of surface proteins. One mutant strain showed fimbriae under electron microscopy. The absence of fimbriae in the other mutant strain was related to a missing 35 kDa membrane protein. Both mutants reacted positively with absorbed anti-31A antiserum suggesting that fímbrial epithopes were expressed. Loss of bovine RBC hemagglutination did not altered the adhesion patter to HeLa cells (AD), either in the fimbriated or in the non-fimbriated mutant strain. Strain BZ43 and the fimbriated mutant strain were non-pathogenic to 3 day old mice in the competition assay. The lack of hemagglutination seemed not to interfere with adhesion properties of the mutant strain in this assay / Mestrado / Genetica / Mestre em Ciências Biológicas