Corynebacterium diphtheriae: Identification and characterization of a channel-forming protein in the cell wall

Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, D-97074 Würzburg, Germany, and Institut de Pharmacologie et Biologie de Structurale, Centre National de la Recherche, Scientifique/Université Paul Sabatier (UMR 5089), F-31077 Toulouse Cedex 04, France

^* To whom correspondence should be addressed. Email: roland.benz{at}mail.uni-wuerzburg.de.

	Abstract

The cell wall fraction of the gram-positive, non-toxic Corynebacterium diphtheriae strain C8_r(-) Tox^- (ATCC 11913) contained a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. The protein had an apparent molecular mass of about 66 kDaDa on tricine-containing SDS-PAGE and consists of subunits of about 5 kDa. Single-channel experiments with the purified protein suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 1M KCl. Further single channel analysis suggested that the cell wall channel is wide and water-filled because it has an only small selectivity for cations over anions and its conductance followed the mobility sequence of cations and anions in the aqueous phase. Antibodies raised against PorA, the subunit of the cell wall channel of C. glutamicum, detected both monomers and oligomers of the isolated protein suggesting highly conserved epitopes between the cell wall channel of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by ELISA. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify its gene cdporA within the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein, 43 amino acids long, without leader sequence. cdporA was expressed in a C. glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels as the purified protein CdporA of C. diphtheriae suggesting that the expressed protein is able to complement PorA and PorH deficiency of the C. glutamicum strain. The study represents the first report of a cell wall channel from a pathogenic Corynebacterium.