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Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus
Author(s) -
Santamarta Irene,
PérezRedondo Rosario,
Lorenzana Luis M.,
Martín Juan F.,
Liras Paloma
Publication year - 2005
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2005.04581.x
Subject(s) - streptomyces clavuligerus , biology , biochemistry , mutant , gene cluster , peptide sequence , microbiology and biotechnology , gene , tetr , repressor , clavulanic acid , gene expression , amoxicillin , antibiotics
Summary Cell‐free extracts from Streptomyces clavuligerus , purified by elution from heparin‐agarose with an ARE‐containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE ccaR ). This sequence, located upstream of the ccaR gene, encodes the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus . Sixty‐one nucleotides upstream of brp another ARE sequence (ARE brp ) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp . A brp ‐deleted mutant, S. clavuligerus Δbrp::neo 1, produced 150–300% clavulanic acid and 120–220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Δbrp::neo 1 lacked a high‐mobility band‐shift due to Brp but still showed the slow‐mobility band‐shift observed in the wild‐type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin biosynthesis by its action on ccaR gene expression.