The cell wall of the Gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming protein was identified, by lipid bilayer experiments, in the cell envelope fractions isolated by sucrose-density centrifugations and in organic solvent of whole cells. It was purified to homogeneity by fast-protein liquid chromatography across a Mono-Q column. The pure protein had a rather low molecular mass of about 5 kDa as judged by SDS-PAGE, which suggested that the cell wall channel is formed by a protein oligomer. The monomer has according to partial sequencing no significant homology to known protein sequences. The purified protein formed large ion-permeable channels in lipid bilayer membranes from phosphatidylcholine/phosphatidylserine mixtures with a single-channel conductance of 5.5 nS in 1 M KCl. Experiments with different salts suggested that the cell wall channel of C. glutamicum was highly cation-selective caused by negative charges localized at the channel mouth. The analysis of the single-channel conductance data using the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.2 nm. Channel-forming properties of the cell wall channel of C. glutamicum were compared with those of mycobacteria. These channels share common features because they form large and water-filled channels that contain point net charges.
SummaryA channel-forming protein was identified in cell wall extracts of the Gram-positive, strictly aerobic bacterium Nocardia farcinica. The cell wall porin was purified to homogeneity and had an apparent molecular mass of about 87 kDa on tricine-containing SDS-PAGE. When the 87 kDa protein was boiled for a longer time in sodium dodecylsulphate (SDS) it dissociated into two subunits with molecular masses of about 19 and 23 kDa. The 87 kDa form of the protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine (PC) phosphatidylserine (PS) mixtures by the formation of ion-permeable channels. The channels had on average a single-channel conductance of 3.0 nS in 1 M KCl, 10 mM Tris-HCl, pH 8, and were found to be cation selective. Asymmetric addition of the cell wall porin to lipid bilayer membranes resulted in an asymmetric voltage dependence. The single-channel conductance was only moderately dependent on the bulk aqueous KCl concentration, which indicated point charge effects on the channel properties. The analysis of the single-channel conductance data in different salt solutions using the Renkin correction factor, and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.4-1.6 nm. Channel-forming properties of the cell wall porin of N. farcinica were compared with those of mycobacteria and corynebacteria. The cell wall porins of these members of the order Actinomycetales share common features because they form large and water-filled channels that contain negative point charges.
Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.
The cell wall of the gram-positive Corynebacterium glutamicum was prepared. It contained an ion-permeable channel with a single-channel conductance of about 6 nS in 1 M KCl. The mobility sequence of the ions in the channel is similar to that in the aqueous phase, suggesting that it is a water-filled channel wide enough to allow unhindered diffusion of ions. The results indicate that we have identified the hydrophilic pathway through the mycolic acid layer of C. glutamicum.
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