RESUMEN
The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalUSpn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalUSpn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.
Asunto(s)
Antibacterianos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia-Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.
Asunto(s)
Bacterias/aislamiento & purificación , Agua de Mar/microbiología , Regiones Antárticas , Bacterias/clasificación , Bacterias/genética , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Evolución Molecular , Microbiota , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , RibotipificaciónRESUMEN
Bacterial richness in maritime Antarctica has been poorly described to date. Phylogenetic affiliation of seawater free-living microbial assemblages was studied from three locations near the Argentinean Jubany Station during two Antarctic summers. Sixty 16S RNA cloned sequences were phylogenetically affiliated to Alphaproteobacteria (30/60 clones), Gammaproteobacteria(19/60 clones), Betaproteobacteria and Cytophaga-Flavobacteriia-Bacteroides (CFB), which were (2/60) and (3/60) respectively. Furthermore, six out of 60 clones could not be classified. Both, Alphaproteobacteria and Gammaproteobacteria, showed several endemic and previously undescribed sequences. Moreover, the absence of Cyanobacteria sequences in our samples is remarkable. In conclusion, we are reporting a rich sequence assemblage composed of widely divergent isolates among themselves and distant from the most closely related sequences currently deposited in data banks.
Asunto(s)
Bacterias/aislamiento & purificación , Agua de Mar/microbiología , Regiones Antárticas , Bacterias/clasificación , Bacterias/genética , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Evolución Molecular , Microbiota , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , RibotipificaciónRESUMEN
An engineered horseradish peroxidase isozyme C (HRP C) gene was constructed by the addition of a 6xArg fusion tail to 6xHis-HRP C by the PCR strategy. The 6xHis-6xArg-HRP C cDNA was expressed in the Sf9 insect cell line from Spodoptera frugiperda infected with Autographa californica nuclear polyhedrosis virus. The recombinant peroxidase isoelectric point was 9.5 as judged by isoelectric focusing and was purified directly from the culture medium at day-6 post-infection by cation-exchange chromatography or immobilised metal ion-affinity chromatography. While the former technique gave a yield of 98.5% with a purification factor of 130, the latter gave only a 68% yield with a purification factor of 140. Results obtained provide evidence that the poly-Arg tag is more effective than the poly-His tag for peroxidase purification from a baculovirus expression system.
