title: Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis.
abstract: 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.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Niederweis
authors.forename: S
authors.initials: S
authors.lastname: Ehrt
authors.forename: C
authors.initials: C
authors.lastname: Heinz
authors.forename: U
authors.initials: U
authors.lastname: Klöcker
authors.forename: S
authors.initials: S
authors.lastname: Karosi
authors.forename: K M
authors.initials: KM
authors.lastname: Swiderek
authors.forename: L W
authors.initials: LW
authors.lastname: Riley
authors.forename: R
authors.initials: R
authors.lastname: Benz
mesh_terms: 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
chemical_list: D018272:Porins; D011994:Recombinant Proteins; C121838:mspA protein, Mycobacterium smegmatis