Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis.
Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal significant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.
Niederweis
M
M
Lehrstuhl für Mikrobiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
Ehrt
S
S
Heinz
C
C
Klöcker
U
U
Karosi
S
S
Swiderek
K M
KM
Riley
L W
LW
Benz
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000235:genetics*; D018272:Porins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis
D018272:Porins; D011994:Recombinant Proteins; C121838:mspA protein, Mycobacterium smegmatis