Genomic organization and in vivo characterization of proteolytic activity of FtsH of Mycobacterium smegmatis SN2.
The ftsH gene of Mycobacterium smegmatis SN2 (MsftsH) was cloned from two independent partial genomic DNA libraries and characterized, along with the identification of ephA and folE as the neighbouring upstream and downstream genes respectively. The genomic organization of the MsftsH locus was found to be identical to that of the Mycobacterium tuberculosis ftsH gene (MtftsH) and similar to that of other bacterial genera, but with divergence in the upstream region. The MsftsH gene is 2.3 kb in size and encodes the AAA (ATPases Associated with diverse cellular Activities) family Zn(2+)-metalloprotease FtsH (MsFtsH) of 85 kDa molecular mass. This was demonstrated from the expression of the full-length recombinant gene in Escherichia coli JM109 cells and from the identification of native MsFtsH in M. smegmatis SN2 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 of E. coli and M. smegmatis cells respectively. Expression of MsFtsH protein in E. coli was toxic and resulted in growth arrest and filamentation of cells. The MsftsH gene did not complement lethality of a DeltaftsH3 : : kan mutation in E. coli, but when expressed in E. coli cells, it efficiently degraded conventional FtsH substrates, namely sigma(32) protein and the protein translocase subunit SecY, of E. coli cells.
Anilkumar
Gopalakrishnapillai
G
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Srinivasan
Ramanujam
R
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Ajitkumar
Parthasarathi
P
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
D049069:ATP-Dependent Proteases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D006360:Heat-Shock Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D045726:Metalloproteases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli