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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,
PèrezRedondo Rosario,
Lorenzana Luis M.,
Martìn 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.1365-2958.2005.04681.x
Subject(s) - streptomyces clavuligerus , biology , biochemistry , mutant , tetr , gene , peptide sequence , microbiology and biotechnology , gene cluster , 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 is located upstream of the ccaR gene, encoding 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 a 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 C biosynthesis by its action on ccaR gene expression.