Submitted to: Journal of Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/23/2015
Publication Date: 9/25/2015
Citation: Kovalskaya, N.Y., Foster Frey, J.A., Donovan, D.M., Bauchan, G.R., Hammond, R. 2015. Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants. Journal of Microbiology and Biotechnology. 26:160-170.
Interpretive Summary: The increasing spread of antibiotic-resistant microorganisms is a growing concern for modern animal husbandry, agriculture and medicine, and also has a significant negative impact on the US economy. In this regard, phage-encoded endolysins, that cause bacterial cell lysis, acquired significant attention as antimicrobials due to their high efficiency and mechanism of action. We report the production of a functionally active endolysin in plants that inhibits the growth of certain bacteria. These results will be interesting to scientists who are producing plant toxic proteins in plant tissues and developing strategies to control bacterial diseases.
Technical Abstract: The emergence and spread of antibiotic-resistant pathogens has led to an increased interest in alternative antimicrobial treatments, such as bacteriophage, bacteriophage-encoded peptidoglycan hydrolases (endolysins) and antimicrobial peptides. In our study, the antimicrobial activity of the CP933 endolysin, encoded by the cryptic bacteriophage CP-933P of the gram-negative bacterium Escherichia coli O157:H7 str. EDL933 was investigated. In order to produce the CP933 protein to investigate its antimicrobial activities, we compared the production of the CP933 protein in a prokaryotic expression system and in Nicotiana benthamiana plants. Expression of the cp933 gene in bacterial cells led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of cp933 in tobacco plants using either a Potato virus X (PVX)-based transient expression vector (pP2C2S) or a binary PVX-based vector (pGR107) resulted in death of the apical part of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its N-terminus to an N-terminal signal peptide from the potato proteinase inhibitor I (PPI-I) to direct CP933 to the delta type vacuoles in plant cells. Although the plants producing the fusion CP933 did not exhibit severe toxic effects on plants, the level of expression was low (0.16 mg per g of plant tissue). The antimicrobial activity of the plant-produced CP933 was evaluated against Clavibacter michiganensis subs. sepedonicus and Escherichia coli (BL 21 (DE3)) and showed 21 and 4% inhibition of bacterial growth, respectively. The results of our experiments demonstrated that targeting of proteins to the delta vacuoles is a promising approach for the production of functionally active proteins that exhibit toxicity when expressed in plant cells.