Identification of a cell wall channel of Streptomyces griseus: the channel contains a binding site for streptomycin.
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 microM 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.
Kim
B H
BH
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Andersen
C
C
Benz
R
R
D001665:Binding Sites; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002473:Cell Wall / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D003960:Diaminopimelic Acid / Q000032:analysis; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008055:Lipids / Q000032:analysis; D008564:Membrane Potentials / Q000187:drug effects; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013305:Streptomyces griseus / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013307:Streptomycin / Q000378:metabolism* / Q000494:pharmacology
D008051:Lipid Bilayers; D008055:Lipids; D018272:Porins; D003960:Diaminopimelic Acid; D013307:Streptomycin