Discovery of a novel channel-forming protein in the cell wall of the non-pathogenic Nocardia corynebacteroides

Detergent extracts of whole cells of the Gram-positive, non-pathogenic, str ictly aerobic bacterium Nocardia corynebacteroides contain channel-forming activity. The protein responsible for channel formation was identified usin g lipid bilayer experiments. It was purified to homogeneity and had an appa rent molecular mass of about 134 kDa on SDS-PAGE when it was solubilized at 40 degreesC. When the 134 kDa protein was heated to 100 degreesC for 10 mi n in sample buffer, it dissociated into subunits with a molecular mass of a bout 23 kDa and focused at pr of 4.5 during isoelectric focusing. The pure 134 kDa protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine-phosphatidylserine mixtur es by the formation of ion-permeable channels. The channels had an average single-channel conductance of 5.5 nS in 1 M KCI and were found to be cation -selective. Asymmetric addition of the 134 kDa protein to lipid bilayer mem branes resulted in an asymmetric voltage-dependence. The analysis of the si ngle-channel conductance as a function of cation radii using the Renkin cor rection factor and the effect of negative charges on channel conductance su ggested that the diameter of the cell wall porin is about 1.0 nm. The chann el characteristics of the cell wall channel of N. corynebacteroides were co mpared with those of other members of the mycolata. They share common featu res because they are composed of small molecular mass subunits and form lar ge and water-filled channels. (C) 2000 Elsevier Science B.V. All rights res erved.