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 and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.
Stahl
C
C
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany.
Kubetzko
S
S
Kaps
I
I
Seeber
S
S
Engelhardt
H
H
Niederweis
M
M
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002463:Cell Membrane Permeability; D002473:Cell Wall / Q000378:metabolism*; D002509:Cephaloridine / Q000378:metabolism; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA
C434272:MspC protein, Mycobacterium smegmatis; C434273:MspD protein, Mycobacterium smegmatis; D018272:Porins; D012333:RNA, Messenger; C121838:mspA protein, Mycobacterium smegmatis; D005947:Glucose; D002509:Cephaloridine