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Journal of Bacteriology, November 2007, p. 7709-7719, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00864-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Corynebacterium diphtheriae: Identification and Characterization of a Channel-Forming Protein in the Cell Wall

Bettina Schiffler,^1, Enrico Barth,^1, Mamadou Daffé,² and Roland Benz^1*

Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, D-97074 Würzburg, Germany,¹ Institut de Pharmacologie et Biologie de Structurale, Centre National de la Recherche, Scientifique/Université Paul Sabatier (UMR 5089), F-31077 Toulouse Cedex 04, France²

Received 4 June 2007/ Accepted 10 August 2007

The cell wall fraction of the gram-positive, nontoxic 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 obtained using organic solvents. The protein had an apparent molecular mass of about 66 kDa as determined on Tricine-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and consisted of subunits having a molecular mass 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 1 M KCl. Further single-channel analysis suggested that the cell wall channel is wide and water filled because it has only slight 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 Corynebacterium glutamicum, detected both monomers and oligomers of the isolated protein, suggesting that there are highly conserved epitopes in the cell wall channels of C. diphtheriae and PorA. Localization of the protein on the cell surface was confirmed by an enzyme-linked immunosorbent assay. The prospective homology of PorA with the cell wall channel of C. diphtheriae was used to identify the cell wall channel gene, cdporA, in the known genome of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a protein that is 43 amino acids long and does not have a 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 CdporA protein of C. diphtheriae formed, suggesting that the expressed protein is able to complement the PorA and PorH deficiency of the C. glutamicum strain. The study is the first report of a cell wall channel in a pathogenic Corynebacterium strain.

Published ahead of print on 24 August 2007.

B.S. and E.B. contributed equally to this paper.