Enterocin P is a bacteriocin produced by Enterococcus faecium P13 and related strains. It has a potential as food biopreservative. In order to optimize the use of this bacteriocin in food preservation, it is important to study its synthesis and secretion. Enterocin P is synthesized as a 71 amino acid precursor (preEntP) with a typical N-terminal signal sequence of 21 amino acids long. The presence of this signal sequence suggests that preEntP is secreted through the General Secretion pathway (Sec-pathway) which would be unusual for two reasons :
1) preEntP is a relatively small secreted protein, and
2) many bacteriocins are secreted by dedicated ATP-dependent transport systems and use an atypical signal sequence. The main objective of this project is to establish the mechanism of preEntP secretion. The preEntP structural gene will be cloned, over expressed and purified from Escherichia coli, and alternatively, synthesized in an in vitro transcription/translation system. PreEntP will be used for in vitro processing and translocation assays employing inner membrane vesicles of E. coli, Ent. faecium and Bacillus subtilis. The dependence on the Sec- and YidC pathways and the identity of the processing enzyme will be determined. The results will be verified by in vivo studies of protein export.