RESUMEN
RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.
Asunto(s)
ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , Streptomyces/genética , Streptomyces/metabolismo , Transactivadores/metabolismo , Sitios de Unión , Vías Biosintéticas/genética , Cefamicinas/biosíntesis , Ácido Clavulánico/biosíntesis , Huella de ADN , Electroforesis en Gel Bidimensional , Ensayo de Cambio de Movilidad Electroforética , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Mutagénesis Insercional , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Streptomycetes are very important industrial bacteria, which produce two thirds of all clinically relevant secondary metabolites. Furthermore, they produce large numbers of eukaryotic cell differentiation and apoptosis inducers. Streptomyces is a mycelial soil bacterium characterized by a complex developmental cycle that includes programmed cell death (PCD) phenomena and sporulation in solid cultures. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that there was no differentiation. Recently, novel aspects concerning differentiation during the presporulation phases were described in solid and liquid cultures, as well as in natural soils. In this review, we analyze the status of knowledge regarding the above-named aspects of Streptomyces differentiation and their relationships with secondary metabolite production.