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Updated: Mar 15, 2026
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Published on: June 11, 2015
Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79
Nittaya Khakhum1, Umaporn Yordpratum2, Atcha Boonmee3
1Department of Biochemistry and Melioidosis Research Center, Faculty of Medicine, Khon Kaen University, 123 Mitraparb Road, Khon Kaen, 40002, Thailand.
Abstract:
The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.
Insights
The ST79 bacteriophage endolysin, peptidase M15A, effectively lyses antibiotic-resistant bacteria, including Gram-negative pathogens. This enzyme shows potential for novel antibacterial therapies, especially when engineered or combined with antibiotics.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Bacteriophage ST79 of Burkholderia pseudomallei utilizes endolysin for host lysis.
- Endolysins are peptidoglycan hydrolases crucial for bacteriophage replication.
- Antibiotic resistance necessitates novel therapeutic strategies against bacterial pathogens.
Purpose of the Study:
- To clone, express, and purify the ST79 endolysin (peptidase M15A).
- To evaluate the lytic activity and enzymatic properties of peptidase M15A against pathogenic bacteria.
- To assess the potential of peptidase M15A as a therapeutic agent.
Main Methods:
- Gene cloning, protein expression, and purification of ST79 endolysin.
- Enzymatic assays using Escherichia coli peptidoglycan as substrate.
- Determination of optimal pH, temperature, and effect of metal ions on enzyme activity.
- Testing lytic activity against a panel of Gram-negative and Gram-positive bacteria.
Main Results:
- Purified peptidase M15A (approx. 18 kDa) exhibits metallopeptidase characteristics.
- Enzyme activity is inhibited by Zn(2+).
- Peptidase M15A demonstrates superior lysis efficiency compared to commercial lysozyme.
- Optimal activity observed at alkaline pH (7.5-9.0) and moderate temperatures (25-42 °C).
- Effective lysis of 18 Gram-negative bacteria (with permeabilization) and Enterococcus sp.
- Limited activity against other Gram-positive bacteria.
Conclusions:
- ST79 endolysin (peptidase M15A) possesses potent lytic activity against a broad spectrum of Gram-negative bacteria.
- Its properties suggest potential for developing new antibacterial treatments, particularly for resistant strains.
- Genetic engineering or combination therapy could enhance its efficacy against challenging bacterial infections.
