Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide

• Published in: Journal of biotechnology 2003-01, Vol.100 (1), p.1-12
• Main Authors: Pecota, Douglas C, Osapay, George, Selsted, Michael E, Wood, Thomas K
• Format: Article
• Language: English
• Subjects: Antibiotics; Antimicrobial; Bacteria - classification; Bacteria - drug effects; Bacteria - ultrastructure; Bacterial Physiological Phenomena - drug effects; Bacterial Toxins - chemical synthesis; Bacterial Toxins - metabolism; Bacterial Toxins - toxicity; Biological and medical sciences; Biotechnology; Cell Membrane Permeability - physiology; Cell Survival - drug effects; Drug Resistance, Bacterial - physiology; Electroporation - methods; Escherichia coli Proteins - chemical synthesis; Escherichia coli Proteins - metabolism; Escherichia coli Proteins - toxicity; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Heat-Shock Response - physiology; Hok peptide; Industrial applications and implications. Economical aspects; Post-segregational killing; Production of active biomolecules; Species Specificity
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(02)00240-7

The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy. Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol. Six microorganisms, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, P. putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing. However, electroporation of Hok (200 μg ml −1) into E. coli cells showed a dramatic reduction (100 000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells. Electroporation of Hok into P. putida was also very effective with a 500-fold reduction in electrocompetent cells (100 μg ml −1). Heat shock in the presence of Hok (380 μg ml −1) resulted in a 5-fold reduction in E. coli cells but had no effect on B. subtilis. In addition, three Hok fragments (Hok(1–28), Hok(31–52) and Hok(16–52)) killed cells when electroporated into E. coli at 200 μg ml −1 (over 1000-fold killing for Hok(1–28), 50-fold killing for Hok(16–52) and over 1000-fold killing for Hok(31–52)). E. coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell.