RESUMO
The RNA Degradosome (RNAD) is a multi-enzyme complex, which performs important functions in post-transcriptional regulation in Escherichia coli with the assistance of regulatory sRNAs and the RNA chaperone Hfq. Although the interaction of the canonical RNAD components with RNase E has been extensively studied, the dynamic nature of the interactions in vivo remains largely unknown. In this work, we explored the rearrangements upon glucose stress using fluorescence energy transfer (hetero-FRET). Results revealed differences in the proximity of the canonical components with 1% (55.5 mM) glucose concentration, with the helicase RhlB and the glycolytic enzyme Enolase exhibiting the largest changes to the C-terminus of RNase E, followed by PNPase. We quantified ptsG mRNA decay and SgrS sRNA synthesis as they mediate bacterial adaptation to glucose stress conditions. We propose that once the mRNA degradation is completed, the RhlB, Enolase and PNPase decrease their proximity to the C-terminus of RNase E. Based on the results, we present a model where the canonical components of the RNAD coalesce when the bacteria is under glucose-6-phosphate stress and associate it with RNA decay. Our results demonstrate that FRET is a helpful tool to study conformational rearrangements in enzymatic complexes in bacteria in vivo.
Assuntos
Escherichia coli/metabolismo , Glucose/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Estresse Fisiológico/efeitos dos fármacos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Estresse Fisiológico/genéticaRESUMO
In the Staphylococcus aureus ATCC25923 strain, the flqB mutation in the 5'untranslated region (5'UTR) of the norA gene causes increased norA mRNA expression and high efflux activity (HEA). The involvement of the norA gene 5'UTR in HEA has not been explored in S. epidermidis; therefore, we examined the function of this region in S. epidermidis clinical isolates. The selection of isolates with HEA was performed based on ethidium bromide (EtBr) MIC values and efflux efficiency (EF) using the semi-automated fluorometric method. The function of the 5'UTR was studied by quantifying the levels of norA expression (RT-qPCR) and by identifying 5'UTR mutations by sequence analysis. Only 10 isolates from a total of 165 (6.1%) had HEA (EtBr MIC = 300 µg/ml and EF ranged from 48.4 to 97.2%). Eight of 10 isolates with HEA had the 5'UTR 95ΔG mutation. Isolates carrying the 95ΔG mutation had higher levels of norA expression compared with those that did not. To corroborate that the 95ΔG mutation is involved in HEA, a strain adapted to EtBr was obtained in vitro. This strain also presented the 95ΔG mutation and had a high level of norA expression and EF, indicating that the 95ΔG mutation is important for the HEA phenotype. The 95ΔG mutation produces a different structure in the Shine-Dalgarno region, which may promote better translation of norA mRNA. To our knowledge, this is the first report to demonstrate the participation of the 5'UTR 95ΔG mutation of the norA gene in the HEA phenotype of S. epidermidis isolates. Here, we propose that the efflux of EtBr is caused by an increment in the transcription and/or translation of the norA gene.
Assuntos
Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Deleção de Sequência , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Antibacterianos/farmacologia , Biofilmes , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/patogenicidadeRESUMO
Con la finalidad de evaluar la eficacia de la gammaglobulina intravenosa como profilaxis en recién nacidos pretérminos, se estudiaron entre mayo de 1988 y abril de 1989 46 neonatos de 36 semanas de gestación y de 1550 grs de peso que nacieron en la Maternidad Armando Castilo Plaza de Maracaibo. En 23 de ellos se utilizó gammaglobulina intravenosa (Sandoglobulina) a razon de 500 mgs/kg. peso durante 4 semanas, en los otros 23 que sirvieron de grupo control no se utilizó. Se descartaron en ambos grupos los antecedentes maternos que pudieran predisponer a infecciones tales como ruptura prematura de membrana y trabajo de parto prolongado. Se cuantifico mediante pruebas de inmunodifusión radial la concentración de IgG en las primeras 48 horas de nacido en los dos grupos estudiados. Al grupo que recibió gammaglobulina se le cuantificó nuevamente a las 2,24,96 y 168 horas. Se encontraron valores iniciles de IgG similares en ambos grupos (578 mgs% para el grupo tratado y con gammaglobulina se observó una elevación del valor base de IgG dos horas después de la infusión del preparado alcanzando una concentración de 1205 mgrs%, estos valores comenzaron a descender a partir de las 24 horas para mantenerse a las 168 horas (7 días) en 778 mgrs%. En el grupo no tratado los valores de IgG para este tiempo fueron muy inferiores (480 mgrs%). No hubo muertes por infeccciones en el grupo de prematuros tratados, mientras que hubo 8 muertes (34.7%) en el grupo no tratado. Estos resultados avalan el beneficio de la gammaglobulina intravenosa utilizada en forma profiláctica en recién nacidos prematuros