Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-10-31T18:51:28Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Bruno Cesar do Espirito Santo.pdf: 2235147 bytes, checksum: 5db508200dcf5ab4b625cfd66f7d76d3 (MD5) / Made available in DSpace on 2018-10-31T18:51:28Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Bruno Cesar do Espirito Santo.pdf: 2235147 bytes, checksum: 5db508200dcf5ab4b625cfd66f7d76d3 (MD5) Previous issue date: 2015-02-27 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / O aumento na produção agrícola deve-se, em boa parte, pelo uso intensivo de agroquímicos, os quais assumem papel relevante. Porém, um dos impactos gerados pelo seu uso é a mudança estrutural e populacional das microbiotas do solo, que precisam de alguma forma tolerar esse xenobióticos para subsistir. São questionados se mecanismos de tolerância são selecionados por pressão seletiva sobre fenótipos específicos ou se existem mecanismos plásticos de adaptação, prescindindo de agentes seletivos específicos. O objetivo deste trabalho foi avaliar os mecanismos de adaptação e respostas celulares, particularmente de isoenzimas de catalase em Escherichia coli, ao contato com o herbicida glifosato, sem seleção prévia para esse herbicida. Resultados mostraram que a deleção em katG permitiu uma tolerância a doses mais elevadas do herbicida, e que a linhagem katE responde melhor as espécies reativas de oxigênio. Taxas de crescimento nos tratamentos com doses elevadas do herbicida se mostraram menores em relação ao controle, mostrando o efeito tóxico sobre as células bacterianas. As taxas de peróxido de hidrogênio foram aumentadas nos tratamentos com herbicidas, o que provavelmente contribuiu para a diminuição no crescimento bacteriano. A linhagem katE foi a mais eficiente em responder aos níveis de peróxido de hidrogênio induzidos pelo glifosato. Os níveis de MDA mostraram-se parecidos em tratamentos com 50 x glifosato em katE e em katG, corroborando a efetividade e especificidade da enzima HPI (katG) na fase log. Considerando que a E. coli não apresentou um contato prévio com o glifosato, sendo uma linhagem desenvolvida para estudos de laboratório, e não adaptada ao ambiente, o sistema de defesa encontrado nessas linhagens pode ser considerado como modelo para outras bactérias de solo, amplo, não específico para o glifosato. Um sistema de respostas não específicas como esse podem aumentar o valor adaptativo de bactérias em solos agrícolas, nos quais são aplicadas diferentes espécies químicas de herbicidas em um tempo relativamente curto. / The increase in agricultural production is due, in large part, by the intensive use of agrochemicals, which assume a significant role. However, one of the impacts generated by its use is structural change and population of the microbiota of the soil, they need somehow tolerate this xenobiotics to subsist. They are asked whether tolerance mechanisms are selected by selective pressure on specific phenotypes or if there are plastic coping mechanisms, regardless of especific. The selective agents aim of this study was to evaluate the mechanisms of adaptation and cellular responses, particularly isozymes of catalase in Escherichia coli when contact with the herbicide glyphosate, without prior selection for this herbicid.Results showed that deletion in katG allowed a tolerance to higher doses of the herbicide, and that katE lineage responsive reactive oxygen species. Growth rates in the treatments with high doses of the herbicide were lower compared to the control, showing the toxic effect on cells As hydrogen peroxide rates were increased in the treatments with herbicides, which probably contributed to the decrease in bacterial growth . The katE strain was more efficient to respond to hydrogen peroxide levels induced by glyphosate. MDA levels were shown to be similar in treatments with 50 x glyphosate in katE and katG, confirming the effectiveness and specificity of the HPI enzyme (katG) in log. Considering phase that E. coli showed no previous contact with glyphosate, a strain being developed for laboratory studies, and not adapted to the environment, the defense system found that strains can be considered as a model for other soil bacteria, broad, not specific to glyphosate. A system of non-specific responses as this may increase the adaptive value of bacteria in agricultural soils, which are applied in different chemical species of herbicide in a relatively short time.

Catalase-peroxidase (KatG) of Mycobacterium tuberculosis is a bifunctional heme enzyme that has been shown to play an important role in the activation of a first line drug, isoniazid (INH), used in the treatment of tuberculosis infection. Mutations in the katG gene have been found to be associated with INH resistance. The most commonly encountered mutation is the Ser315Thr point mutation. In this dissertation, the x-ray crystallographic structures of MtbKatG and the mutant enzyme KatG[S315T] are presented to explore the molecular basis of the INH activation and resistance. The structure is dimeric and contains a heme cofactor in each subunit of the dimer. The most important change in KatG[S315T] is due to the presence of the methyl group of the threonine 315 side chain, which is located at the narrowest part of the substrate channel. The protruding methyl group effectively constricts the accessibility to the heme by closing down the dimensions of the channel, constraining the substrate entrance. VirB11 of Brucella abortus is a hexameric ATPase that belongs to the type IV secretion system. The crystal structure of BaVirB11 was found to contain six molecules per asymmetric unit. The Walker A (P loop), His box, and Glu box from the C-terminal domain are located at the interface of the N- and C-terminal domain. A large conformational change was found in the linker region when compared with that of HP0525 structure, the VirB11 analogous from H. pylori. To elucidate the functional role of each domain, seven functional mutations were generated and used for biochemical studies. The GER motif and the linker region were found to be crucial for ATP hydrolysis activity of BaVirB11. Mutations in the GER motif (R101Q) and the linker region (R120E) of BaVirB11 completely abolish the ATP hydrolysis activity of the enzyme. The binding affinities of the two mutants to the ATP; however, are similar to that of the wild-type enzyme, indicating that mutation in the GER motif or the linker region has no effect on ATP binding.

Made available in DSpace on 2014-07-29T15:30:37Z (GMT). No. of bitstreams: 1 Dissertacao_sueli.pdf: 1117407 bytes, checksum: d50c0b1dad4e594bc8cd5d3880aadcab (MD5) Previous issue date: 2010-03-11 / Multidrug-resistant tuberculosis is a challenge worldwide. Rapid diagnosis by molecular techniques can provide a more aggressive and appropriate initial therapy. This study aimed to analyze the molecular basis of resistance to isoniazid (INH) and rifampin (R) of Mycobacterium tuberculosis strains isolated from cases of human tuberculosis in Goiás and to genetically determine the causes of the observed resistances. Of the 4.607 cultures for mycobacteria processed in the period of September of 2005 and December of 2007, 24 isolates from 16 patients resistant to at least H and/or R were analyzed. We compared the results obtained by phenotypic tests with mutations in key genes responsible for the development of resistance to these drugs, the rpoB gene for isolates resistant to R and katG gene for strains resistant to H. Seventy one percent of the isolates were resistant to H, and the mutations involved with resistance observed in the katG gene were in codon 315 (41%). The most frequent mutations observed in the rpoB gene of the R resistant isolates (71%) were in codons 456 (76.5%) and 451 (17.6%). Our findings are similar to those reported in the literature. We conclude that the percentage of agreement between genotypic and phenotypic tests was 41% for H and 94% for R considering the number of isolates and 40% and 91%, respectively considering the number of patients. / A tuberculose multidroga resistente representa um desafio em escala mundial. O diagnóstico rápido através de técnicas moleculares é capaz de proporcionar uma terapêutica inicial mais agressiva e adequada. Este trabalho teve como objetivo analisar as bases moleculares da resistência à isoniazida (H) e rifampicina (R) de cepas de Mycobacterium tuberculosis isoladas de casos de tuberculose em Goiás e determinar geneticamente as causas destas resistências. Do total de 4.607 culturas para micobactérias realizadas no período de setembro 2005 a dezembro de 2007, foram analisados 24 isolados de 16 pacientes resistentes a H e/ou R. Os resultados obtidos dos testes fenotípicos de sensibilidade aos antimicrobianos foram comparados às mutações observadas nos principais genes responsáveis pelo desenvolvimento de resistência a estas drogas, gene rpoB para isolados resistentes à R e gene katG para os isolados resistentes à H. Dentre os 24 isolados, 71% eram fenotípicamente resistentes a H e as únicas mutações envolvidas com resistência foram observadas no códon 315 (41%). Dos isolados resistentes a R (71%), foram observadas mutações nos códons 456 (76,5%), 451 (17,6%) e 447 (5,9%). Nossos achados estão em concordância com as principais mutações observadas nos isolados resistentes a R e/ou H descritos na literatura. O percentual de concordância entre os testes fenotípicos e genotípicos foi de 41% para H e 94% para R considerando o número de isolados e de 40% e 91% respectivamente considerando-se o número de pacientes.

Catalase-peroxidases (KatGs), responsible for the activation of the anti-tubercular prodrug isoniazid (INH), are unusual members of the class I plant peroxidase family that possess strong catalase activity as well as peroxidase activity. Due to their strong catalase activity and their ability to activate INH, KatGs have been the subject of intense study for many years, and thus the goal of this work is to further characterize this enzyme in the hope of gaining a better understanding into these unusual reactions. Recent successful crystallization of a few representative KatGs revealed a unique covalent Met-Tyr-Trp cross-link joined to the conserved tryptophan in the heme active site, along with a nearby arginine that is in ionic association with the cross-linked tyrosine. Using the KatG from Burkholderia pseudomallei (BpKatG) as a model, site-directed mutagenesis to these residues revealed that they were essential for catalase, but not peroxidase activity. Structural and kinetic analysis revealed that Arg426 acts as a molecular switch, moving between 2 conformations, favoring heme oxidation when not in association with Tyr238 and favoring heme reduction when in association with Tyr238 by imparting its influence on the heme through the cross-link. Analysis of the reaction with peroxyacetic acid using stopped-flow spectrophotometry revealed an initial, rapidly formed enzyme-substrate complex before the formation of the oxoferryl compound I. Kinetic characterization revealed that formation of both the enzyme-substrate complex and the oxoferryl species were dependent on peroxyacetic acid concentration implying that 2 molecules of peroxyacetic acid are required to form the oxoferryl compound I intermediate. Successful co-crystallization with INH and its co-substrate, NAD+ has revealed their binding sites for the first time in a KatG. The NAD+ binding site is 20 Å from the entrance to the heme cavity, involving interactions primarily with the ADP portion of the molecule. The best defined INH binding site is located in a funnel shaped channel on the opposite side of the protein from the entrance channel that requires the movement of a glutamate residue for binding. The structures suggest that once INH is cleaved to the isonicotinoyl radical it diffuses to the NAD+ binding site to form the final active antimicrobial compound, IN-NAD, in a non-enzymatic reaction enhanced by the enzyme’s ability to bind NAD+.

Catalase-peroxidases (KatGs), responsible for the activation of the anti-tubercular prodrug isoniazid (INH), are unusual members of the class I plant peroxidase family that possess strong catalase activity as well as peroxidase activity. Due to their strong catalase activity and their ability to activate INH, KatGs have been the subject of intense study for many years, and thus the goal of this work is to further characterize this enzyme in the hope of gaining a better understanding into these unusual reactions. Recent successful crystallization of a few representative KatGs revealed a unique covalent Met-Tyr-Trp cross-link joined to the conserved tryptophan in the heme active site, along with a nearby arginine that is in ionic association with the cross-linked tyrosine. Using the KatG from Burkholderia pseudomallei (BpKatG) as a model, site-directed mutagenesis to these residues revealed that they were essential for catalase, but not peroxidase activity. Structural and kinetic analysis revealed that Arg426 acts as a molecular switch, moving between 2 conformations, favoring heme oxidation when not in association with Tyr238 and favoring heme reduction when in association with Tyr238 by imparting its influence on the heme through the cross-link. Analysis of the reaction with peroxyacetic acid using stopped-flow spectrophotometry revealed an initial, rapidly formed enzyme-substrate complex before the formation of the oxoferryl compound I. Kinetic characterization revealed that formation of both the enzyme-substrate complex and the oxoferryl species were dependent on peroxyacetic acid concentration implying that 2 molecules of peroxyacetic acid are required to form the oxoferryl compound I intermediate. Successful co-crystallization with INH and its co-substrate, NAD+ has revealed their binding sites for the first time in a KatG. The NAD+ binding site is 20 Å from the entrance to the heme cavity, involving interactions primarily with the ADP portion of the molecule. The best defined INH binding site is located in a funnel shaped channel on the opposite side of the protein from the entrance channel that requires the movement of a glutamate residue for binding. The structures suggest that once INH is cleaved to the isonicotinoyl radical it diffuses to the NAD+ binding site to form the final active antimicrobial compound, IN-NAD, in a non-enzymatic reaction enhanced by the enzyme’s ability to bind NAD+.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.