MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy a nd an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable am ounts of mspA mRNA, as revealed by amplification using mspA-specific primer s and reverse-transcribed RNA. Detergent extracts of the Delta mspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smeg matis. The chromosome of M. smegmatis encodes three proteins very similar t o MspA, Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the Delta mspA mutant. The properties of thi s porin, such as single channel conductance, extreme stability against dena turation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA, Deletion of mspA reduced the cell wall permeability towar ds cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molec ules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the Delta mspA mutant. The minimal permeability coefficient o f the Delta mspA mutant for glucose was 7.2 x 10(-8) cm s(-1), which is the lowest value reported so far for bacteria. This is the first experimental evidence that porins are the major determinants of the exceptionally low pe rmeability of mycobacteria to hydrophilic molecules.