Identification of a cell-wall channel in the corynemycolic acid-free Gram-positive bacterium Corynebacterium amycolatum.

Abstract

As part of a comparative study of the cell wall of corynebacteria, a channel-forming protein was characterized in Corynebacterium amycolatum, a species devoid of corynemycolic acids. Corynebacterium amycolatum cells were disrupted and the cell envelope subjected to two different separation procedures, differential centrifugation to separate the different fractions of the cell envelope, and sucrose-step-gradient density centrifugation. The fractions obtained by the two methods were analyzed for lipid composition, NADH oxidase activity, and the formation of ion-permeable channels in lipid bilayers. High channel-forming activity was always detected in fractions expected to contain only cell-wall components. The highest NADH-oxidase activity was found in other fractions, indicating that the cell-wall fraction was distinct from the membrane fraction. The cell wall was found to contain an ion-permeable channel with a single-channel conductance of about 3.8 nS in 1 M KCl. The channel-forming protein, with an apparent molecular mass of 45 kDa, was purified to homogeneity using FPLC and preparative SDS-PAGE. Single-channel experiments suggested that the cell-wall channel is wide and water-filled and has a narrow selectivity for cations. Analysis of the fatty-acid composition of extractable lipids and delipidated cells suggested that the cell wall of C. amycolatum contains enough lipids to form an additional permeability barrier on the surface of the bacteria, thus accounting for the presence of the cell-wall channel.