Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.

This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.

Magíster en Bioquímica área de Especialización en Bioquímica ambiental / Memoria para optar al Título de Bioquímico / Los problemas ambientales y la disminución de las reservas de petróleo han reiniciado la búsqueda de combustibles alternativos a los combustibles fósiles. El etanol se presenta como una alternativa sustentable para resolver esta problemática. En la naturaleza existen microorganismos silvestres productores de etanol, tales como la bacteria Zymomonas mobilis y la levadura Saccharomyces cerevisiae. Si bien estos dos microorganismos son altamente eficientes en la generación de etanol, sólo son capaces de utilizar azúcares de 6 carbonos, limitando la producción industrial de etanol solo a materias primas que contengan hexosas. En los residuos lignocelulósicos se encuentra una gran reserva de hidratos de carbono almacenada como polímeros de glucosa (celulosa) o azúcares de 5 carbonos como xilosa y arabinosa. Para el aprovechamiento de pentosas disponibles en esta materia prima se han modificado estos microorganismos, mediante la introducción de los genes de las enzimas necesarias para metabolizar arabinosa y xilosa o se han incorporado los genes de las enzimas de la ruta etanológica de Z. mobilis, piruvato descarboxilasa (pdc) y alcohol deshidrogenasa II (adhB) a bacterias capaces de utilizar pentosas, como Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis y Bacillus subtilis. El género Pseudomonas, principalmente Pseudomonas aeruginosa, posee muchas de las características necesarias para la producción industrial de etanol, es una versátil bacteria Gram negativo, capaz de adaptarse y sobrevivir bajo amplio rango de condiciones ambientales y al igual que Z. mobilis asimila azúcares por la ruta de Entner-Doudoroff, además posee una toleracia a etanol mayor que bacterias E. coli recombinantes utilizadas en la producción de etanol. El objetivo general de este trabajo es la construcción de un operón productor de etanol para la expresión de los genes pdc y adhB de la ruta etanologénica de Z. mobilis en P. aeruginosa PAO1. El diseño del operón artificial incluyó los siguientes elementos genéticos: el promotor del operón inducible arabinosa (PBAD), sitio de unión a ribosoma (RBS), gen pdc, gen adhB y un terminador transcripcional para P. aeruginosa. Las primeras etapas para la construcción del operon pet, contemplaron la realización de 2 construcciones genéticas mediante PCR. La primera comprende la mínima región codificante del gen pdc y una región adaptadora de 33 pb (adp), la segunda corresponde a la unión de adp2 (secuencia que contiene una zona complementaria a adp y un sitio de unión a ribosoma) con la mínima región codificante del gen adhB y un terminador transcripcional específico para Pseudomonas. Los análisis de restricción, PCR y secuenciación mostraron la obtención de un fragmento 1740 pb correspondiente al producto pdc-adp, un fragmento de 1247 pb para el producto adp2- adhB-term. Para los productos de PCR de la fusión de las construcciones pdc-adp con adp2- adhB-term se obtuvieron amplicones inespecíficos de tamaños superiores e inferiores al tamaño teórico esperado (3 Kb). De manera alternativa para la obtención de la construcción que contenga los genes pdc y adhB con la secuencia adaptadora y de unión a ribosoma, se realizaron reacciones de ligación de ambos fragmentos utilizando la enzima T4 DNA ligasa, las cuales fueron clonadas en el vector pSC-B. De todos los clones analizados solo uno contenía la construcción esperada (plasmidio pCL27-B), pero los análisis de PCR revelaron que los genes no se encontraban en la orientación adecuada, por lo tanto no fue posible obtener la construcción del fragmento pdc-adp-RBS-adhB, construcción clave en el término de la construcción del operón pet. / Environmental problems and declining oil reserves have resumed the search for alternative fuels to fossil fuels. Ethanol is presented as a sustainable alternative to solve this problem. In nature there are wild ethanol producing microorganisms, such as bacteria Zymomonas mobilis and yeast Saccharomyces cerevisiae. While these two microorganisms are highly efficient in the generation of ethanol, are capable of using only 6 carbon sugars, limiting the industrial production of ethanol only to raw material containing hexoses. In lignocellulosic waste there is a large reservoir of stored carbohydrates like glucose polymers (cellulose) or 5-carbon sugars such as xylose and arabinose. For the utilization of available pentoses in this raw material, such microorganisms have been modified by the introduction of genes of the necessary enzymes to metabolize arabinose and xylose or enzymes genes have been incorporated of etanologic pathway from Z. mobilis, pyruvate decarboxylase (pdc) and alcohol deshidrogenasa II (adhB) to bacteria capable to use pentoses, such as Escherichia coli, Klebsiella oxycota, Lactobacillus plantarum, Lactococcus lactis and Bacillus subtilis. The genus Pseudomonas, mainly Pseudomonas aeruginosa, has many of the features needed for industrial production of ethanol, is a versatile Gram negative bacteria, able to adapt and survive under wide range of environmental conditions and like Z. mobilis assimilated sugars by the Entner-Doudoroff pathway, and in addition has ethanol higher tolerance than recombined E. coli bacteria used in the production of ethanol. The overall aim of this work is the construction of an ethanol producer operon for the expression of pdc and adhB genes from the Z. mobilis etanologic pathway on P. aeruginosa PAO1. The artificial operon design included the following genetic elements: the arabinose inducible operon promoter (PBAD), ribosome binding site (RBS), pdc gene, adhB gene and a transcriptional terminator to P. aeruginosa. The first step to construct the pet operon, contemplated 2 genetic constructs by PCR. The first involves a pdc gene minimum coding region and a adapter region of 33 bp (adp), the second corresponds to the union of adp2 (sequence that contains a complementary zone to adp and a ribosome binding site) with adhB gene minimal encoding region and a specific transcriptional terminator to Pseudomonas. The restriction analysis, PCR and sequencing showed the obtention of a 1740 pb fragment corresponding to the pdc-adp product, a 1247 pb fragment for the product adp2-adhB-term. For the PCR products of pdc-adp and adp2-adhB-term construction fusion, non specific amplicons were obtained in higher and lower sizes than the theoretical size expected (3 Kb). Alternatively for the obtention of the construction which have the pdc and adhB genes with the adapter sequence and ribosome binding, ligation reactions were performed of both fragments using T4 DNA ligase enzyme, which were cloned on the vector pSC- B. Of all the clones analyzed just one contained the expected construction (plasmid pCL27-B), but PCR analysis revealed that genes were not in the proper orientation, therefore it was not possible to obtain the construction of fragment pdc-adp-RBS-adhB, key construction in the completion of the pet operon construction.

Thesis (M.S. )--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains 155 p. Includes bibliographical references p. 151-152.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves [207]-216).

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xv, 205 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 163-190).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains ix, 132 p.; also includes graphics (some col.). Includes bibliographical references (p. 112-132). Available online via OhioLINK's ETD Center

Orientador: Marcelo de Carvalho Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T01:23:41Z (GMT). No. of bitstreams: 1 Cacci_LucianaCamila_M.pdf: 1733904 bytes, checksum: 2616f843895d251e8e236cdd873fe436 (MD5) Previous issue date: 2007 / Resumo: Introdução: As metalo beta-lactamases, presentes em Pseudomonas aeruginosa assim como em diversos microrganismos Gram negativos, são grande ameaça ao tratamento dos pacientes portadores de infecções causadas por este microrganismo. Objetivos: A produção de MBLs e a relação genética foram investigadas em isolados de P. aeruginosa resistentes ao Imipenem, recuperados de infecções hospitalares. Descrição do estudo: Estudo restropectivo em uma amostra de microrganismos. O estudo foi conduzido em dois hospitais universitários, em Campinas. Todos os isolados de P. aeruginosa resistentes ao Imipenem foram coletados de pacientes hospitalizados no período de Março de 2000 a Dezembro de 2004. Métodos: O método da disco-difusão foi utilizado para confirmar a resistência ao Imipenem. O E-test MBL@ foi feito para verificar a produção de MBLs e a Concentração Inibitória Mínima (CIM) do Imipenem. Os ftagmentos das seqüências dos genes blaIMP-I, blaVIM-I, blaVIM-2 e blaSPM-I foram amplificados. Resultados: Cento e vinte e oito isolados resistentes ao Imipenem foram coletados durante o período do estudo. A maioria dos isolados exibiu CIM do Imipenem maior ou igual a 256 ug/mL. A análise por macrorestrição com a enzima SpeI através do Pulsed Field Gel Electrophoresis (PFGE) mostrou um polimorfismo significativo. Apenas 15 cepas puderam ser distribuídas em sete "clusters", seis com dois isolados e um com três isolados. Noventa e oito isolados resistentes ao Imipenem foram triados para a pesquisa da produção de MBL. Setenta isolados apresentaram produção de MBL e o fragmento do gene blaSPM-l pôde ser amplificado em 12 isolados. Nas cepas restantes nenhum outro tipo de MBL referente aos genes blaIMP-l, blaVIM-I, blaVIM-2 foi encontrado. Conclusão: A disseminação de cepas de P. aeruginosa produtoras de MEL genotipicamente heterogêneas foi documentada nos hospitais estudados. Apenas a metalo beta-lactamase SPM-1 foi encontrada entre essas cepas / Abstract: Objective: Genetic relatedness and Metallo-lactamase production was investigated in Imipenem resistant Pseudomonas aeruginosa recovered from hospital acquired infections. Design: Descriptive study in a convenient sample of organisms. Setting: Two 400-bed tertiary care teaching hospitals, in Campinas, Brazil. All Imipenem resistant P. aeruginosa, recovered from March, 2000 through December 2004 from hospitalized patients were collected Methods: Disk diffusion tests were used to confirm Imipenem resistance. E-test MBL@ was done to check for MBL production, ando Imipenem MIC's blasPM-l, .blaIMP-l, blaVIM-l and blaVIM-2 sequences were amplified. Results: A sample of 128 Imipenem resistant P. aeruginosa isolates was collected during the study period. Most isolates exhibited Imipenem MIC's > 256 ug/mL. Macrorestriction analysis (Spel) using pulsed field gel electrophoresis (PFGE) showed a substantial polymorphism. Only 15 strains could be allocated to seven clusters, six with two isolates and one with three isolates. Ninety-eight Imipenem resistant isolates were screened for MBL production. Seventy isolates showed MBL production, and blasPM-l sequence could be amplified from 12 isolates. In the remaining strains no other MBL-type from blaIMP-l, blaVIM-l and blaVIM-2 investigated in the study was demonstrated. Conclusion: Dissemination of MBL producing-genotypically heterogenous Pseudomonas aeruginosa strains was documented in the hospitals studied. Only SPM-l metallo-_-lactamase was found among these strains / Mestrado / Ciencias Basicas / Mestre em Clinica Medica