The cell wall of Micromonospora purpurea contains a high conductance channel.
In this communication it is demonstrated that the cell wall of the gram-positive bacterium Micromonospora purpurea contains a cell wall channel for the passage of hydrophilic solutes. The channel-forming protein was identified in sucrose step-density-gradient fractions of the cell envelope and in whole cell extracts using either organic solvent or detergent and the lipid bilayer technique. The fractions of the sucrose step-density centrifugation were assayed for NADH-oxidase activity and for the formation of ion-permeable channels in lipid bilayers. The highest NADH-oxidase activity and the highest channel-forming ability were found in different fractions. The cell wall fraction was identified by the presence of meso-diaminopimelic acid and contained an ion-permeable channel with the extremely high single-channel conductance of about 14 nS in 1 M KCl. The channel-forming unit was purified to homogeneity by FPLC on a HiTrap-Q column. It was identified as a heat- and SDS-resistant 200-kDa band on SDS-PAGE and formed the same general diffusion pores in lipid bilayer membranes as those formed by detergent extracts of the cell wall fraction of the sucrose step-density centrifugation. The channels were slightly selective for potassium ions over chloride, possibly caused by an excess of negative charges in or near the channel.
Kim
Bong-Hui
BH
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
Wengerter
Karin
K
Benz
Roland
R
D002473:Cell Wall / Q000737:chemistry*; D003960:Diaminopimelic Acid / Q000302:isolation & purification; D004553:Electric Conductivity; D007473:Ion Channels / Q000302:isolation & purification*; D008051:Lipid Bilayers; D008847:Micromonospora / Q000737:chemistry*; D010539:Permeability
D007473:Ion Channels; D008051:Lipid Bilayers; D003960:Diaminopimelic Acid