title: Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.
abstract: 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.
authors.affiliation: INBIOTEC, Instituto de Biotecnología de León, Avda. Real n°.1, Parque Científico de León, 24006 León, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Santamarta
authors.forename: M T
authors.initials: MT
authors.lastname: López-García
authors.forename: A
authors.initials: A
authors.lastname: Kurt
authors.forename: N
authors.initials: N
authors.lastname: Nárdiz
authors.forename: R
authors.initials: R
authors.lastname: Alvarez-Álvarez
authors.forename: R
authors.initials: R
authors.lastname: Pérez-Redondo
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
authors.forename: P
authors.initials: P
authors.lastname: Liras
mesh_terms: D001665:Binding Sites; D053898:Biosynthetic Pathways / Q000235:genetics; D002513:Cephamycins / Q000096:biosynthesis; D019818:Clavulanic Acid / Q000096:biosynthesis; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D002513:Cephamycins; D004269:DNA, Bacterial; D012333:RNA, Messenger; D015534:Trans-Activators; D019818:Clavulanic Acid