CHARACTERIZATION AND PRODUCTION OF LYSIN FROM MET P1-301 BACTERIOPHAGE FOR BIOCONTROL APPROACHES AGAINST PATHOGENIC ESCHERICHIA COLI

Date

2024-6-24

Author

Yücefaydalı, Ayşenur

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Today, one of the most common foodborne pathogens is E. coli, which is Gram negative, rod shaped, non-spore forming bacteria. However, the increasing antibiotic resistance of E. coli has been reported worldwide. For this reason, new searches for alternatives to antibiotics have begun. It was thought that bacteriophages which are viruses that infect and lyse living bacterial cells could be an alternative to antibiotics. Bacteriophages use their lytic enzymes, called lysins, to lyse the host cell. Lysins are produced during intracellular proliferation of phages and cause cell death by disrupting the integrity of the host cell wall. Besides, unlike the bacteriophages, lysins act quickly since they do not need to bacterial replication. Moreover, they target the conserved areas of cell wall therefore the possibility of resistance development by bacteria against lysins are low. In this study, the genome of bacteriophage MET P1-301 against E. coli O104:H4 was whole genome sequenced using both Illumina and Oxford Nanopore Technologies. A putative lysin from bacteriophage MET P1-301, LysEc301, belonging to the lysozyme-like superfamily, was cloned, produced, and characterized. Recombinant LysEc301 (rLysEc301) is made up of a single domain with a molecular weight of approximately 17 kDa. The yield of rLysEc301 was around 85 ng/µL. In Tris-HCl buffer, the bactericidal activity of rLysEc301 against E. coli O104:H4, E. coli O157:H7, EPEC, ETEC and Salmonella Typhimurium was determined. It was observed that rLysEc301 alone reduced both E. coli O104:H4 and E. coli O157:H7 concentration by 3 log(CFU/mL) and Salmonella Typhimurium concentration by 2.5 log(CFU/mL). Besides, combination of rLysEc301 and EDTA reduced the concentration of both EPEC and ETEC by approximately 2.5 log(CFU/mL). Further analysis should be performed to investigate the stability of rLysEc301 in potential food applications.

Subject Keywords

Phage-derived lysin, E. coli, Biocontrol, Recombinant protein

URI

https://hdl.handle.net/11511/110134

Collections

Graduate School of Natural and Applied Sciences, Thesis