Study of the properties of a channel-forming protein of the cell wall of the gram-positive bacterium Mycobacterium phlei

The gram-positive bacterium Mycobacterium phlei was treated with detergents . Reconstitution experiments using lipid bilayers suggested that the deterg ent extracts contain a channel forming protein. The protein was purified to homogeneity by preparative SDS-PAGE and identified as a protein with an ap parent molecular mass of about 135 kDa. The channel-forming unit dissociate d into subunits with a molecular mass of about 22 kDa when it was boiled in 80% dimethylsulfoxid (DMSO). The channel has on average a single channel c onductance of 4.5 nS in 1 M KCl and is highly voltage-dependent in an asymm etric fashion when the protein is added to only one side of the membrane. Z ero-current membrane potential measurements with different salts implied th at the channel is highly cation-selective because of negative point charges in or near the channel mouth. Analysis of the single-channel conductance a s a function of the hydrated cation radii using the Renkin correction facto r and the effect of the negative point charges on the single-channel conduc tance suggest that the diameter of the cell wall channel is about 1.8 to 2. 0 nm. The channel properties were compared with those of other members of t he mycolata and suggest that these channels share common features. Southern blots demonstrated that the chromosome of M. phlei and other mycolata test ed contain homologous sequences to mspA (gene of the cell wall porin of Myc obacterium smegmatis).