Identification and characterization of the two-component cell lysis cassette encoded by temperate bacteriophageϕPYB5 ofLactobacillus fermentum

Abstract

Aims: To characterize the two‐component cell lysis cassette comprised of holin (Hyb5) and endolysin (Lyb5) encoded by Lactobacillus fermentum temperate bacteriophage ϕPYB5, and illustrate the potential application of Lyb5 as therapeutic agents. Methods and Results: The hyb5–lyb5 cassette was cloned from the genome library of ϕPYB5, and the hyb5, lyb5 and hyb5–lyb5 cassette were expressed in E. coli BL21, respectively. The molecular weight of Hyb5 indicated by SDS‐PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage of β‐galactosidase. However, the Hyb5–Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, Lyb5 exhibited a broad lytic spectrum. Conclusions: Overexpression of hyb5, lyb5 and hyb5–lyb5 cassette were carried out in E. coli and Lyb5 exhibited a broad lytic spectrum. Significance and Impact of the Study: The Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram‐positive strains including Staphylococcus aureus as well as Gram‐negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.

Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

“…The endolysin gene lyb5 from Lb. fermentum temperate bacteriophage PYB5 encodes a protein of 418 amino acids with a deduced molecular mass of 45 kDa (30). A signal peptide was predicted by using SOPMA software, and the cleavage site was located between amino acids 29 and 30 at the N terminus of Lyb5 (Fig.…”

“…casei cells. At different time points (15,30,60,120,180, and 240 min) after being mixed with GFP-Ly5C, cells were pelleted and washed with PBS and the fluorescence level per OD 600 unit was measured.…”

“…Endolysins from bacteriophages are cell wall hydrolases involved in cell lysis to release the progeny particles from the host cells (9,30). Most endolysins lack a signal peptide and are translocated across the membrane by the aid of the holin protein.…”

“…The sequence of the endolysin gene lyb5 from the genome of PYB5 has been deposited in GenBank under accession number EF531306, and the gene product has been successfully expressed in Escherichia coli and has shown a broad lytic spectrum (30).…”

Molecular Dissection of Phage Endolysin

“…Earlier studies with lysins have utilized chloroform to make the plasma membrane permeable that then allows the diffusion of lysins to the cell wall peptidoglycan and its digestion (53)(54)(55). Several other studies have followed the lysin effect after its co-expression with Holin (25,56). Our methodology developed here will not only help in studying the activity of these proteins in detail but will also aid in identifying new proteins from other bacteriophages when this method of expression is followed in a suitable host.…”