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Open AccessArticle

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

Department of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland
Department of Virology, Medicum, University of Helsinki, 00290 Helsinki, Finland
Division Animal and Human Health Engineering, Kasteelpark Arenberg 21 - box 2462, 3001 Leuven, Belgium
Laboratory of Microbiology, Wageningen University and Research, 6708 WE Wageningen, The Netherlands
Division of Clinical Microbiology, Helsinki University Hospital, HUSLAB, 00290 Helsinki, Finland
Author to whom correspondence should be addressed.
Present address: Department of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands.
Viruses 2019, 11(11), 1057;
Received: 26 September 2019 / Revised: 29 October 2019 / Accepted: 12 November 2019 / Published: 14 November 2019
(This article belongs to the Section Bacterial Viruses)
The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts. View Full-Text
Keywords: HPUF; antibacterials; screening; bacteriophages; assay; toxic; φR1-RT HPUF; antibacterials; screening; bacteriophages; assay; toxic; φR1-RT
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Mohanraj, U.; Wan, X.; Spruit, C.M.; Skurnik, M.; Pajunen, M.I. A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins. Viruses 2019, 11, 1057.

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