Corynebacterium diphtheriae : Identification and 1 characterization of a channel - forming protein in the cell 2 wall 3

1 The cell wall fraction of the gram-positive, non-toxic Corynebacterium diphtheriae strain 2 C8r(-) Tox (ATCC 11913) contained a channel-forming protein as judged from reconstitution 3 experiments with artificial lipid bilayer experiments. The channel-forming protein was present 4 in detergent treated cell walls and in extracts of whole cells using organic solvents. The 5 protein had an apparent molecular mass of about 66 kDaDa on tricine-containing SDS-PAGE 6 and consists of subunits of about 5 kDa. Single-channel experiments with the purified protein 7 suggested that the protein formed channels with a single-channel conductance of 2.25 nS in 8 1M KCl. Further single channel analysis suggested that the cell wall channel is wide and 9 water-filled because it has an only small selectivity for cations over anions and its 10 conductance followed the mobility sequence of cations and anions in the aqueous phase. 11 Antibodies raised against PorA, the subunit of the cell wall channel of C. glutamicum, 12 detected both monomers and oligomers of the isolated protein suggesting highly conserved 13 epitopes between the cell wall channel of C. diphtheriae and PorA. Localization of the protein 14 on the cell surface was confirmed by ELISA. The prospective homology of PorA with the cell 15 wall channel of C. diphtheriae was used to identify its gene cdporA within the known genome 16 of C. diphtheriae. The gene and its flanking regions were cloned and sequenced. CdporA is a 17 protein, 43 amino acids long, without leader sequence. cdporA was expressed in a C. 18 glutamicum strain that lacked the major outer membrane channels PorA and PorH. Organic 19 solvent extracts of the transformed cells formed in lipid bilayer membranes the same channels 20 as the purified protein CdporA of C. diphtheriae suggesting that the expressed protein is able 21 to complement PorA and PorH deficiency of the C. glutamicum strain. The study represents 22 the first report of a cell wall channel from a pathogenic Corynebacterium. 23 A CE PT ED on N ovem er 3, 2017 by gest http/jb.asm .rg/ D ow nladed fom