Functional analysis of the N-terminal region of endolysin Lyb5 encoded by Lactobacillus fermentum bacteriophage φPYB5

• Endolysin Lyb5 of phage φPYB5 was exported by the N-terminal putative signal peptide.
• The N-terminal region of endolysin Lyb5 acted as a functional signal–anchor–release (SAR) domain.
• The SAR domain was experimentally identified for the first time in LAB phages.
• The SAR domain might be generally employed for endolysin export in phages.

Lactobacillus fermentum temperate bacteriophage φPYB5 uses endolysin Lyb5 and holin Hyb5 to burst the host cell. Previous results showed that expression of Lyb5 in Escherichia coli caused host cell lysis slowly, leading us to suppose that Lyb5 could pass the cytoplasmic membrane partly. In this work, the function of a putative signal peptide (SPLyb5) at the N-terminal of Lyb5 was investigated. In E. coli, the cell adopted a spherical shape during induction of Lyb5 protein, while morphological changes were not observed during expression of the SPLyb5 truncation, indicating that the SPLyb5 motif may serve as a functional signal peptide. However, SPLyb5 was not proteolytically cleaved at the predicted site during the translocation of Lyb5, and the expressed Lyb5 protein appeared in the cytoplasm, cytoplasmic membrane and periplasm fractions with the same molecular mass. Similar results were obtained using Lactococcus lactis as a host to express Lyb5. These results indicated that SPLyb5 could direct Lyb5 to the periplasm in a membrane-tethered form, and then release it as a soluble active enzyme into the periplasm. In addition, SPLyb5 could also drive the fused NucleaseB protein to the extracytoplasm environment in E. coli as well as in L. lactis. We proposed that in Gram-negative and Gram-positive hosts SPLyb5 acted as a signal–anchor–release domain, which was firstly identified here by experimental evidences in lactic acid bacteria phages. The application of signal–anchor–release domain for endolysin export in bacteriophages infecting Gram-positive and Gram-negative hosts was discussed.