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Functioning of the stable signal peptide of the pCloDF13-encoded bacteriocin release protein
J Luirink1, B Duim, J W de Gier
1Department of Molecular Microbiology, Faculty of Biology, Vrije Universiteit, Amsterdam, The Netherlands.
Insights
The stable signal peptide of bacteriocin release protein (BRP) is crucial for releasing cloacin DF13. Replacing it with an unstable peptide prevented cloacin release, highlighting its role in translocation.
Area of Science:
- Molecular Biology
- Bacteriology
- Protein Biochemistry
Background:
- Bacteriocin release protein (BRP) is a lipoprotein essential for releasing bacteriocins.
- BRP is synthesized with a unique N-terminal signal peptide that remains stable after cleavage.
Purpose of the Study:
- To investigate the function of the stable BRP signal peptide.
- To determine the signal peptide's role in cloacin DF13 release, cell lysis, and periplasmic protein leakage.
Main Methods:
- Genetic engineering to replace the BRP signal peptide with the unstable murein lipoprotein (Lpp) signal peptide.
- Analysis of protein acylation and processing by signal peptidase II.
- Assessing cloacin DF13 release, cell lysis, and periplasmic enzyme activity.
Main Results:
- The hybrid protein, lacking a stable signal peptide, was correctly processed.
- Specific release of cloacin DF13 was abolished with the hybrid protein.
- Cell lysis and periplasmic enzyme release remained unaffected.
Conclusions:
- The stable BRP signal peptide plays a critical role in the translocation of cloacin DF13 across the cytoplasmic membrane.
- The signal peptide's stability, not just its presence, is key for specific bacteriocin release.
Abstract:
The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.