Studies of the subtilin leader peptide as a translocation signal in Escherichia coli K12.
Subtilin is an antimicrobial peptide of the lantibiotic family that is produced by Gram-positive Bacillus subtilis, and its biosynthesis involves expression of presubtilin which consists of a leader segment and a mature segment. The leader segment is unlike a typical sec-type general secretion signal, and its ability to mediate translocation through a non-sec pathway has been previously studied by fusing the subtilin leader to an alkaline phosphatase reporter and expressing it in B. subtilis 168 [Izaguirre, G. and Hansen, J. N. (1997) Appl. Environ. Microbiol. 63, 3965-3971]. In this work, we have expressed the same subtilin leader-AP fusion in Gram-negative Escherichia coli, and found that the AP polypeptide is translocated into the periplasmic compartment and assembles into an enzymatically active form. The subtilin leader segment was not cleaved from this enzymatically active AP, which remained associated with the membrane. Conversion of the cells to spheroplasts followed by treatment with proteinase K showed that about 50% of the bound AP was sufficiently exposed on the surface of the spheroplasts to be inactivated by proteolytic cleavage.
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
L K
LK
Paul
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Izaguirre
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JN
Hansen
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001430:Bacteriocins; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli; D010455:Peptides; D010957:Plasmids / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000378:metabolism*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000469:Alkaline Phosphatase; C002836:subtilin