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Identification of a cell wall channel of Streptomyces griseus : the channel contains a binding site for streptomycin
Author(s) -
Kim BongHui,
Andersen Christian,
Benz Roland
Publication year - 2001
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 - 10.1046/j.1365-2958.2001.02544.x
Subject(s) - streptomyces griseus , biology , cell wall , centrifugation , cell envelope , chromatography , streptomycin , biochemistry , biophysics , chemistry , streptomyces , bacteria , escherichia coli , antibiotics , gene , genetics
Cells of the Gram‐positive actinomycete Streptomyces griseus were disrupted and the cell envelope was subjected to sucrose step‐gradient centrifugation. The different fractions were analysed for NADH‐oxidase activity and the formation of ion‐permeable channels in lipid bilayers. Highest channel‐forming activity and highest NADH‐oxidase activity were found in different fractions. The cell wall fraction contained an ion‐permeable channel with a single‐channel conductance of 850 pS in 1 M KCl. The channel‐forming protein, with an apparent molecular mass of 28 kDa, was purified to homogeneity using fast protein liquid chromatography after the extraction of whole cells with detergent. Single‐channel experiments suggest that the cell wall channel is wide and water‐filled. Titration experiments with streptomycin produced by S. griseus suggested that the cell wall channel binds this antibiotic with a half saturation constant of about 6 mM in 1 M KCl. The binding of streptomycin was found to be ionic strength dependent and the half saturation constant decreased to 60 µM at 0.1 M KCl. The results indicate that the 28 kDa protein represents the hydrophilic pathway through the cell wall of the Gram‐positive bacterium S. griseus .