Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2

Abstract

TheftsHgene ofMycobacterium smegmatisSN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification ofephAandfolEas the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsHlocus was found to be identical to that of theMycobacterium tuberculosis ftsHgene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsHgene is 2·3 kb in size and encodes the AAA (ATPasesAssociated with diverse cellularActivities) family Zn2+-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene inEscherichia coliJM109 cells and from the identification of native MsFtsH inM. smegmatisSN2 cell lysates by Western blotting with anti-MtFtsH and anti-EcFtsH antibodies respectively. The recombinant and the native MsFtsH proteins were found localized to the membrane ofE. coliandM. smegmatiscells respectively. Expression of MsFtsH protein inE. coliwas toxic and resulted in growth arrest and filamentation of cells. The MsftsHgene did not complement lethality of a ΔftsH3 : : kan mutation inE. coli, but when expressed inE. colicells, it efficiently degraded conventional FtsH substrates, namelyσ32protein and the protein translocase subunit SecY, ofE. colicells.