RESUMEN
Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Hierro , Metaloproteínas/genética , Rhodobacter capsulatus/genética , Superóxido Dismutasa/genética , Aerobiosis/genética , Secuencia de Aminoácidos , Clonación Molecular , Resistencia a Medicamentos , Regulación Bacteriana de la Expresión Génica , Genes Esenciales , Prueba de Complementación Genética , Datos de Secuencia Molecular , Oxidantes/farmacología , Estrés Oxidativo , Rhodobacter capsulatus/enzimología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Superóxidos/farmacologíaRESUMEN
Membranes from in vivo labeled cells of Rhodobacter capsulatus U43[pTX35] grown photosynthetically carried 60% of the [32P]-Pi in the "heavy" fraction (HM) after sucrose gradient sedimentation. Metal-chelating chromatography of either"heavy" or "light" (LM) membrane fractions rendered similar Bchl-protein complex profiles after octyl-glucoside treatment,including most of the radioactivity in the same corresponding elution fraction (F II). Similar labeling distribution of pigment-protein complexes was obtained for membranes of dark-grown cells induced by lowering oxygen tension. Fractions derived from HM showed highly labeled LHIalpha, whereas the same complex from LM was essentially [32P]-Pi-free, as revealed by SDS-PAGE followed by autoradiography. Phospholipid analysis showed a similar pattern for membranes isolated from cells photosynthetically or semiaerobically grown, being the most abundant: phosphatidylglycerol,phosphatidylethanolamine, cardiolipin, and phosphatidylcholine. Part of the phospholipids from HM comigrated with LHIalpha during SDS-PAGE and dissociated from the complexes only after solvent extraction and hydrophobic chromatography. However, a small amount remained always attached to LHIalpha,indicating an unusual strong interaction. These results suggest the existence of two operationally defined membrane regions carrying LHIalpha complexes differing in phosphorylation status and protein-phospholipid interaction.
Asunto(s)
Proteínas Bacterianas/análisis , Fosfolípidos/análisis , Rhodobacter capsulatus/química , Autorradiografía , Electroforesis en Gel de Poliacrilamida , Mutación , FotosíntesisRESUMEN
Apresentando o universo e a metodologia adotada durante a pesquisa, o artigo identifica como fatores determinantes da pesquisa: uso de drogas, práticas sexuais de risco, a escolha de métodos contraceptivos, riscos envolvidos pela gravidez, discriminação social, baixa estima, violência. Em seguida, apresenta alguns dos mitos e conceitos errados relacionados com as formas de infecção por HIV e os métodos de prevenção