Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from Rhodococcus erythropolis

Abstract

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.

Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis

“…Rhodococcus has mycolic acids in the size range C 30 -C 54 , and Nocardia in the size range C 42 -C 66 but in both cases with no functional groups in the mero-chain other than the possibility of one or more double bonds [5,6,7,8] .…”

Rhodococcus equi

“…It forms a permeability barrier to hydrophilic compounds, resulting in the formation of a periplasmic space [9,13,63]. Obviously this has consequences for the import of small hydrophilic molecules, which is highlighted by the presence of porins which have been identified in the cell walls of Mycobacterium, Corynebacterium, Rhodococcus and Nocardia [42,45,46,58]. The mycolic acid containing cell wall is likely to be of importance for survival of these bacteria under harsh enviromental conditions as occur for example within macrophages.…”

Identification and characterization of a cell wall porin from <i>Gordonia jacobaea</i>

“…Anion selective channel-forming proteins have only been found in C. glutamicum, C. efficiens, M. bovis and R. equi. (Hünten et al, 2005a, b;Lichtinger et al, 1998Lichtinger et al, , 2000Rieß et al, 2003).…”

“…In our work, we were not able to identify any signal peptide in the c6/294 protein by using the SignalP algorithm. Other porin-like proteins lacking signal peptides have been described in the gram-positive bacterial group mycolata, such as the small pore-forming peptides PorA and PorH of C. glutamicum, which means that these peptides are exported to the cell wall by a yet unknown mechanism (Hünten et al, 2005a, b;Lichtinger et al, 2000). Similarly, the poreforming protein OmpAtb from M. tuberculosis does not show a signal peptide, probably because its N-terminus does not adequately fit the definition of classical signal peptides (Alahari et al, 2007;Senaratne et al, 1998).…”