Discovery of a cell wall porin in the mycolic‐acid‐containing actinomycete Dietzia marisDSM 43672

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Abstract

The cell wall of the Gram-positive mycolic-acid-containing actinomycete Dietzia maris DSM 43672 was found to contain a pore-forming protein, as observed from reconstitution experiments with artificial lipid bilayer experiments in the presence of cell wall extracts. The cell wall porin was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 120 kDa on tricine-containing SDS/PAGE. The 120 kDa protein dissociated into subunits with a molecular mass of about 35 kDa when it was heated to 100 °C in 8 m urea. The 120 kDa protein, here named PorADm , formed ion-permeable channels in lipid bilayer membranes with a high single-channel conductance of about 5.8 nS in 1 m KCl. Asymmetric addition of PorADm to lipid bilayer membranes resulted in an asymmetric voltage dependence. Zero-current membrane potential measurements with different salt solutions suggested that the porin of D. maris is cation-selective because of negative charges localized at the channel mouth. Analysis of the single-channel conductance using non-electrolytes with known hydrodynamic radii indicated that the diameter of the cell wall channel is about 2 nm. The channel characteristics of the cell wall porin of D. maris are compared with those of other members of the mycolata. They share some common features because they are composed of small molecular mass subunits and form large and water-filled channels. The porin was subjected to protein analysis by mass spectrometry but its sequence had no significant homology to any known porin sequences.

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