Univ., Diplomarbeit, --Zugl.: Kaiserslautern, 1981.

Zugl.: Kaiserslautern, Univ., Diss., 1987

Zugl.: Kaiserslautern, Univ., Diplomarbeit, 1981

Ureases são metaloenzimas dependentes de níquel, amplamente distribuída em bactérias, fungos e plantas. Estas enzimas atuam na catálise da hidrólise da ureia a amônia e dióxido de carbono. Proteus mirabilis é uma bactéria patogênica, produtora de urease, um de seus mais importantes fatores de virulência. Esta bactéria Gram-negativa se comporta como um uropatógeno oportunista responsável por severas infecções em pacientes hospitalizados. A amônia liberada pela hidrólise da ureia catalisada pela urease de Proteus mirabilis (PMU) causa um aumento no pH levando à formação de microclima, possibilitando a colonização do patógeno no trato urinário do hospedeiro. A PMU apresenta alta similaridade com outras ureases, como a urease de sementes de “Jack bean” (JBU) e a urease de Helicobacter pylori (HPU), para as quais nosso grupo descreveu diversas atividades biológicas que são independentes da hidrólise de ureia. Neste trabalho, nós produzimos PMU, e logo depois investigamos se esta, assim como a JBU e a HPU, apresenta atividades não relacionadas à atividade enzimática. As condições de cultivo para expressão da PMU expressa em Escherichia coli HB101 foram otimizadas pela metodologia de superfície de resposta. Concentrações de níquel, ureia e tempos de indução foram testados. A purificação da enzima recombinante foi obtida em 3 etapas cromatográficas. A primeira, uma HiTrapQTM HP (pH 7,5) onde a urease foi eluida com 400 mMol.L-1 de KCl. O pico das frações eluídas foram reunidas, dialisadas e aplicadas na coluna HiLoad 26/10 Q-SepharoseTM HP, usando o mesmo tampão e sal para eluição. As frações ativas foram novamente reunidas e a PMU foi submetida a cromatografia de gel filtração (Superdex 200TM 26/60-pg). A PMU apresenta estabilidade na faixa de pH 7,0 a 8,5, com seu pH ótimo estimado em 8,0. Alta atividade ureolítica pode ser detectada de 37 oC a 48 oC. Diferentes soluções salinas induzem o aumento na atividade enzimática desta urease, e quanto maior o tempo de exposição, maior a tendência a este aumento. Assim como a JBU, esta urease é capaz de inibir o crescimento de leveduras, mas diferentemente desta e da HPU, a PMU não apresenta atividade inibitória sobre a germinação de esporos e o crescimento de fungos filamentosos. As ureases de P. mirabilis e de H. pylori apresentam regiões de semelhança com o peptídeo proveniente do colágeno, e de acordo com testes de modelagem, esta região estaria exposta para interação com receptores localizados nas membranas de plaquetas, visto que ambas ativam plaquetas resultando na formação de agregados. / Ureases are Ni-dependent metalloenzymes, widespread in bacteria, fungi and plants, that catalyze the hydrolysis of urea into ammonium and carbon dioxide. The pathogenic bacteria Proteus mirabilis produces urease as virulence factor. Proteus mirabilis is a Gram negative opportunistic uropathogen, which causes severe infections in hospitalized patients. Ammonia released from urea hydrolysis by Proteus mirabilis urease (PMU) increases the local pH and forms a microclimate which allows the colonization of the host urinary tract. PMU presents high similarity to other ureases, such as that from Jack bean seeds (JBU) or from Helicobacter pylori (HPU), for which our group has described biological activities unrelated to urea hydrolysis. Here we aimed to investigate whether PMU shares with JBU and HPU other properties unrelated to enzyme activity. Growth conditions of PMU-expressing Escherichia coli HB101 were optimized by response surface methodology prior to purification. Concentrations of nickel, urea, and induction time were tested. A partially purified recombinant enzyme was obtained after 3 chromatographic steps. In the first, a HiTrapQTM HP (pH 7.5), urease eluted with 400 mMol.L-1 KCl. Peak fractions were pooled, dialyzed and loaded in a HiLoad 26/10 Q-SepharoseTM HP column using same buffer and eluting salt. The active fractions were pooled and PMU was submitted to gel filtration (Superdex 200TM26/60-pg). The enzyme was stable in the range of pH 7.0 up to 8.5, with optimum pH at 8.0. The ureolitic activity is high from 37 oC up to 48 oC. Different salts increased the ureolytic activity of PMU, the longer the exposition, the higher was the increase in activity. PMU inhibited yeast growth, similarly to the effect induced by JBU. Differently from JBU and HPU, this urease did not inhibit spore germination and growth of different filamentous fungi. Ureases from P. mirabilis and H. pylori presented regions of homology with collagen, and according to modeling tests, these region are exposed to receptor recognition localized in platelets membrane, which might explain their platelet aggregating effect.

Ureases são metaloenzimas dependentes de níquel, amplamente distribuída em bactérias, fungos e plantas. Estas enzimas atuam na catálise da hidrólise da ureia a amônia e dióxido de carbono. Proteus mirabilis é uma bactéria patogênica, produtora de urease, um de seus mais importantes fatores de virulência. Esta bactéria Gram-negativa se comporta como um uropatógeno oportunista responsável por severas infecções em pacientes hospitalizados. A amônia liberada pela hidrólise da ureia catalisada pela urease de Proteus mirabilis (PMU) causa um aumento no pH levando à formação de microclima, possibilitando a colonização do patógeno no trato urinário do hospedeiro. A PMU apresenta alta similaridade com outras ureases, como a urease de sementes de “Jack bean” (JBU) e a urease de Helicobacter pylori (HPU), para as quais nosso grupo descreveu diversas atividades biológicas que são independentes da hidrólise de ureia. Neste trabalho, nós produzimos PMU, e logo depois investigamos se esta, assim como a JBU e a HPU, apresenta atividades não relacionadas à atividade enzimática. As condições de cultivo para expressão da PMU expressa em Escherichia coli HB101 foram otimizadas pela metodologia de superfície de resposta. Concentrações de níquel, ureia e tempos de indução foram testados. A purificação da enzima recombinante foi obtida em 3 etapas cromatográficas. A primeira, uma HiTrapQTM HP (pH 7,5) onde a urease foi eluida com 400 mMol.L-1 de KCl. O pico das frações eluídas foram reunidas, dialisadas e aplicadas na coluna HiLoad 26/10 Q-SepharoseTM HP, usando o mesmo tampão e sal para eluição. As frações ativas foram novamente reunidas e a PMU foi submetida a cromatografia de gel filtração (Superdex 200TM 26/60-pg). A PMU apresenta estabilidade na faixa de pH 7,0 a 8,5, com seu pH ótimo estimado em 8,0. Alta atividade ureolítica pode ser detectada de 37 oC a 48 oC. Diferentes soluções salinas induzem o aumento na atividade enzimática desta urease, e quanto maior o tempo de exposição, maior a tendência a este aumento. Assim como a JBU, esta urease é capaz de inibir o crescimento de leveduras, mas diferentemente desta e da HPU, a PMU não apresenta atividade inibitória sobre a germinação de esporos e o crescimento de fungos filamentosos. As ureases de P. mirabilis e de H. pylori apresentam regiões de semelhança com o peptídeo proveniente do colágeno, e de acordo com testes de modelagem, esta região estaria exposta para interação com receptores localizados nas membranas de plaquetas, visto que ambas ativam plaquetas resultando na formação de agregados. / Ureases are Ni-dependent metalloenzymes, widespread in bacteria, fungi and plants, that catalyze the hydrolysis of urea into ammonium and carbon dioxide. The pathogenic bacteria Proteus mirabilis produces urease as virulence factor. Proteus mirabilis is a Gram negative opportunistic uropathogen, which causes severe infections in hospitalized patients. Ammonia released from urea hydrolysis by Proteus mirabilis urease (PMU) increases the local pH and forms a microclimate which allows the colonization of the host urinary tract. PMU presents high similarity to other ureases, such as that from Jack bean seeds (JBU) or from Helicobacter pylori (HPU), for which our group has described biological activities unrelated to urea hydrolysis. Here we aimed to investigate whether PMU shares with JBU and HPU other properties unrelated to enzyme activity. Growth conditions of PMU-expressing Escherichia coli HB101 were optimized by response surface methodology prior to purification. Concentrations of nickel, urea, and induction time were tested. A partially purified recombinant enzyme was obtained after 3 chromatographic steps. In the first, a HiTrapQTM HP (pH 7.5), urease eluted with 400 mMol.L-1 KCl. Peak fractions were pooled, dialyzed and loaded in a HiLoad 26/10 Q-SepharoseTM HP column using same buffer and eluting salt. The active fractions were pooled and PMU was submitted to gel filtration (Superdex 200TM26/60-pg). The enzyme was stable in the range of pH 7.0 up to 8.5, with optimum pH at 8.0. The ureolitic activity is high from 37 oC up to 48 oC. Different salts increased the ureolytic activity of PMU, the longer the exposition, the higher was the increase in activity. PMU inhibited yeast growth, similarly to the effect induced by JBU. Differently from JBU and HPU, this urease did not inhibit spore germination and growth of different filamentous fungi. Ureases from P. mirabilis and H. pylori presented regions of homology with collagen, and according to modeling tests, these region are exposed to receptor recognition localized in platelets membrane, which might explain their platelet aggregating effect.

Ureases são metaloenzimas dependentes de níquel, amplamente distribuída em bactérias, fungos e plantas. Estas enzimas atuam na catálise da hidrólise da ureia a amônia e dióxido de carbono. Proteus mirabilis é uma bactéria patogênica, produtora de urease, um de seus mais importantes fatores de virulência. Esta bactéria Gram-negativa se comporta como um uropatógeno oportunista responsável por severas infecções em pacientes hospitalizados. A amônia liberada pela hidrólise da ureia catalisada pela urease de Proteus mirabilis (PMU) causa um aumento no pH levando à formação de microclima, possibilitando a colonização do patógeno no trato urinário do hospedeiro. A PMU apresenta alta similaridade com outras ureases, como a urease de sementes de “Jack bean” (JBU) e a urease de Helicobacter pylori (HPU), para as quais nosso grupo descreveu diversas atividades biológicas que são independentes da hidrólise de ureia. Neste trabalho, nós produzimos PMU, e logo depois investigamos se esta, assim como a JBU e a HPU, apresenta atividades não relacionadas à atividade enzimática. As condições de cultivo para expressão da PMU expressa em Escherichia coli HB101 foram otimizadas pela metodologia de superfície de resposta. Concentrações de níquel, ureia e tempos de indução foram testados. A purificação da enzima recombinante foi obtida em 3 etapas cromatográficas. A primeira, uma HiTrapQTM HP (pH 7,5) onde a urease foi eluida com 400 mMol.L-1 de KCl. O pico das frações eluídas foram reunidas, dialisadas e aplicadas na coluna HiLoad 26/10 Q-SepharoseTM HP, usando o mesmo tampão e sal para eluição. As frações ativas foram novamente reunidas e a PMU foi submetida a cromatografia de gel filtração (Superdex 200TM 26/60-pg). A PMU apresenta estabilidade na faixa de pH 7,0 a 8,5, com seu pH ótimo estimado em 8,0. Alta atividade ureolítica pode ser detectada de 37 oC a 48 oC. Diferentes soluções salinas induzem o aumento na atividade enzimática desta urease, e quanto maior o tempo de exposição, maior a tendência a este aumento. Assim como a JBU, esta urease é capaz de inibir o crescimento de leveduras, mas diferentemente desta e da HPU, a PMU não apresenta atividade inibitória sobre a germinação de esporos e o crescimento de fungos filamentosos. As ureases de P. mirabilis e de H. pylori apresentam regiões de semelhança com o peptídeo proveniente do colágeno, e de acordo com testes de modelagem, esta região estaria exposta para interação com receptores localizados nas membranas de plaquetas, visto que ambas ativam plaquetas resultando na formação de agregados. / Ureases are Ni-dependent metalloenzymes, widespread in bacteria, fungi and plants, that catalyze the hydrolysis of urea into ammonium and carbon dioxide. The pathogenic bacteria Proteus mirabilis produces urease as virulence factor. Proteus mirabilis is a Gram negative opportunistic uropathogen, which causes severe infections in hospitalized patients. Ammonia released from urea hydrolysis by Proteus mirabilis urease (PMU) increases the local pH and forms a microclimate which allows the colonization of the host urinary tract. PMU presents high similarity to other ureases, such as that from Jack bean seeds (JBU) or from Helicobacter pylori (HPU), for which our group has described biological activities unrelated to urea hydrolysis. Here we aimed to investigate whether PMU shares with JBU and HPU other properties unrelated to enzyme activity. Growth conditions of PMU-expressing Escherichia coli HB101 were optimized by response surface methodology prior to purification. Concentrations of nickel, urea, and induction time were tested. A partially purified recombinant enzyme was obtained after 3 chromatographic steps. In the first, a HiTrapQTM HP (pH 7.5), urease eluted with 400 mMol.L-1 KCl. Peak fractions were pooled, dialyzed and loaded in a HiLoad 26/10 Q-SepharoseTM HP column using same buffer and eluting salt. The active fractions were pooled and PMU was submitted to gel filtration (Superdex 200TM26/60-pg). The enzyme was stable in the range of pH 7.0 up to 8.5, with optimum pH at 8.0. The ureolitic activity is high from 37 oC up to 48 oC. Different salts increased the ureolytic activity of PMU, the longer the exposition, the higher was the increase in activity. PMU inhibited yeast growth, similarly to the effect induced by JBU. Differently from JBU and HPU, this urease did not inhibit spore germination and growth of different filamentous fungi. Ureases from P. mirabilis and H. pylori presented regions of homology with collagen, and according to modeling tests, these region are exposed to receptor recognition localized in platelets membrane, which might explain their platelet aggregating effect.

The nature of the symbiotic association of the red alga Harveyella mirabilis (Reinsch) Schmitz and Reinke (Cryptonemiales) and its red algal hosts Odonthalia and Rhodomela (Ceremiales) was investigated. The distribution of H. mirabilis was revised to include additional host species as well as a greater range of occurrence in the northeast Pacific. A study of procarp development confirms that H. mirabilis should be retained in the order Cryptonemiales. Based only upon morphological criteria, H. mirabilis has been defined previously as parasitic on its red algal hosts. In the present study, a new definition of parasitism has been formulated to include physiological as well as morphological aspects of parasitism. Accordingly, a red algal parasite is defined as any red alga living temporarily or permanently within or on a host, deriving benefits from it and causing it harm. The association of Harveyella mirabilis and its hosts is considered with regard to (1) the reproductive and developmental dependence of H. mirabilis on a specific host, (2) the possibility of metabolite exchange between host and Harveyella, and (3) the effects of the presence of Harveyella on its host. A field and laboratory study of the development and reproduction of H. mirabilis has revealed that the completion of its life history is dependent on the presence of a suitable host and that reproduction and development are affected by seasonal changes in environmental parameters. Initial spore germination occurs in host wounds inflicted primarily by grazing isopods and amphipods. Rhizoidal cells penetrate the walls between host cells and establish secondary pit connections with host cells. Subsequent development is characterized by rapid proliferation of the rhizoidal cells within the host, a rupturing of the host's outer wall region and the final development of a colourless reproductive pustule. Morphological and cytological studies have shown that H. mirabilis conforms to a typical "Polysiphonia-type" life history. The effects of seasonal fluctuations in seawater salinity and temperature, and changes in the hours of bright sunshine (photoperiod) on the reproduction and development of H. mirabilis have been examined over a 20-month period. Gametogenesis occurred in northeast Pacific populations in the spring and fall between a seawater temperature range of 8.5-11 C whereas tetrasporogenesis occurred in the late winter as both photoperiod and water temperature increased. H. mirabilis is physiologically dependent upon the host 0. floccosa as a source of nutrients. Liquid scintillation analysis and light microscopic autoradiography have demonstrated that H¹⁴CO₃⁻ is photosynthetically assimilated by the host and subsequently transferred to H. mirabilis. The primary flow occurs from host medullary cells to adjacent rhizoidal cells of H. mirabilis. A secondary transfer occurs from host cells dispersed in the pustule to adjacent H. mirabilis cells. Ion exchange chromatography and chemical extraction techniques were employed to separate labeled fractions of 0. floccosa and H. mirabilis after various periods of ¹⁴C-translocation. The change in radioactivity in the alcohol-soluble neutral fraction most closely paralleled the total increase in radioactivity in H. mirabilis and the corresponding decrease in 0. floccosa. To identify translocated compounds, the labeled neutral fractions were separated by paper chromatography. An increase in radioactivity was associated with an unknown substance in Harveyella which had an R glucose value similar to glucuronic and galacturonic acids in both acidic and basic solvent systems. A concomitant decrease in radioactivity was associated with both a high R glucose unknown and mannitol in 0. floccosa neutral sugars separated in both basic and acidic solvents. Cortical, medullary, and rhizoidal cells of H. mirabilis were examined by light and electron microscopy to determine the structural mechanisms involved in nutrient transfer. A membrane system in the rhizoidal cells, consisting of the plasmalemma, pinocytotic vesicles, multivesicular and concentric bodies, ER, dictyosomes, microbodies and an extensive vacuolar system may be involved in the uptake, processing and distribution of nutrients throughout these cells. Histochemical identification was made of proteins, lipids and carbohydrates associated with this vacuolar/vesicle system. Light autoradiography, supported this proposed membrane uptake mechanism. Plasma-lemmal extensions (plasmalemmavilli) of H. mirabilis medullary cells in the pustule may also be involved in nutrient uptake. The effects of H. mirabilis on the host 0. floccosa were also examined by light and electron microscopy. In host medullary cells adjacent to rhizoidal cells, changes occur in vacuolation, plasmalemmal vesiculation, ER, plastids, nuclei, pit connections and walls. Direct penetration of host cells by rhizoidal cells of H. mirabilis occasionally occurs resulting in death of the host cells. Host medullary and cortical cells dispersed in the emergent pustule show few of the degenerative responses noted in host cells adjacent to H. mirabilis, rhizoidal cells. On the contrary, host cell division and photosynthetic assimilation of H¹⁴CO₃⁻ all increase. Spherical virus-like solitary bodies (S-bodies) occur in all cells of H. mirabilis and in host cells attached to H. mirabilis by secondary pit connections. The possibility that these structures may induce the infective host response is discussed. In the concluding discussion the possible evolution of H. mirabilis considered in relation to proposed theories of the origin of red algal parasites. / Science, Faculty of / Botany, Department of / Graduate

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Bacterial biofilms form on liquid/air and liquid/solid surfaces and consist of cells combined with an extracellular matrix such as exopolysaccharides, extracellular DNA, and glycoproteins. Bacteria have up to a 1000-fold increase of antibiotic resistance in biofilms compared to planktonic cells. Furthermore, biofilm cells show better tolerance to adverse environmental conditions such as nutrition limitations, temperature changes, pH changes, and non-optimal osmotic conditions. In Escherichia coli, the outer membrane protein OmpA increased biofilm formation on polystyrene, polypropylene, and polyvinyl chloride surfaces while it decreased biofilm formation on glass surfaces. This surface-dependent phenotype was because OmpA inhibits cellulose production by inducing the CpxRA two-component signal transduction pathway, and cellulose inhibits biofilm formation on plastic due to its hydrophilic nature. We discovered, and then engineered, BdcA (formerly YjgI), for biofilm dispersal. We found that in E. coli, BdcA increases motility and extracellular DNA production while it decreases exopolysaccharide production, cell length, and aggregation. We reasoned that the 3, 5-cyclic diguanylic acid (c-di-GMP) levels increase upon deleting bdcA, and showed that BdcA binds c-di-GMP in vitro. In addition, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete biofilm dispersal. We isolated Proteus mirabilis from the blowfly Lucilia sericata, which swarmed significantly. By motility screening and complementation with putative interkingdom signal molecules that have been shown to attract flies, we found lactic acid, phenol, NaOH, KOH, putrescine, and ammonia restore the swarming motility of seven different swarming deficient mutants. These mutants and putative signal molecules will be further tested for fly attraction and oviposition. Acetylation of lysine residues is conserved in all three kingdoms although its role in bacteria is not clear. We demonstrated that acetylation enables E. coli to withstand environmental stresses. Specifically, the bacteria became more resistant to heat and oxidative stress. Furthermore, we showed that the increase in oxidative stress resistance is due to the induction of catalase gene katG. Hence we demonstrate for the first time a specific physiological role for acetylation in prokaryotes.

Thesis (doctoral)--Rijks-Universitet te Groningen, 1917. / Includes bibliographical references.