A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, Bacteriophages and Culture Conditions
2.2. Mass Spectrometry (MS) Data Analysis of Phage Proteins
2.3. Comparative Protein Analysis of Hypothetical Proteins of Unknown Function (HPUFs) and Genome Annotation
2.4. PCR Amplification of Control and Hypothetical Proteins of Unknown Function Genes for Cloning
2.5. Cloning
2.6. Screening for Toxic Hypothetical Proteins of Unknown Function Genes
2.7. Confirmation of Protein Toxicity
2.8. Structure Prediction of Toxic HPUFs
2.9. Statistics
3. Results
3.1. Elimination of Hypothetical Proteins of Unknown Function (HPUFs) by LC-MS/MS Analysis of Phage Particle Proteomes
3.2. Further Elimination of Hypothetical Proteins of Unknown Function (HPUFs) by BLASTp and HHpred Analyses
3.3. Ligations of Toxic and Non-Toxic Control Genes Show Differences in Plating Efficiency
3.4. Eight Potential Toxic Protein Hits Identified from an Initial CFU-Based Screening Approach
3.5. Toxic Protein Hits Gp064, Gp136, Gp137 and Gp232 Inhibit the Growth of E. coli Cells
3.6. Modelling Studies
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name | Description | Source |
---|---|---|
E. coli strains | ||
DH10B | Used for HPUFs screening | NEB, USA |
DH5α | Used for pBAD30 cloning | NEB, USA |
Bacteriophages | ||
ϕR1-RT | Used as a template for amplifying all the ϕR1-RT encoded genes used in the current study | [11] |
T4 | Used as a template for amplifying regB and ndd control genes | [14] |
Plasmids | ||
pETSmt3- DCD-1L | Used as template to amplify the dcd-1l control gene | [15] |
pU11L4 | This plasmid consists of pUC19 with a KpnI-PstI linker in the MCS and luxAB genes under expression of ompF promoter of YeO:3 at the SapI site (Figure S1. | This study |
ϕX174 RF1 | Used as template to amplify the lysE control gene | Thermo Fisher Scientific, USA |
pBAD30 | Plasmid with arabinose-inducible promoter to express ϕR1-RT HPUFs in E. coli for confirmatory assay | [16] |
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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. https://doi.org/10.3390/v11111057
Mohanraj U, Wan X, Spruit CM, Skurnik M, Pajunen MI. A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins. Viruses. 2019; 11(11):1057. https://doi.org/10.3390/v11111057
Chicago/Turabian StyleMohanraj, Ushanandini, Xing Wan, Cindy M. Spruit, Mikael Skurnik, and Maria I. Pajunen. 2019. "A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins" Viruses 11, no. 11: 1057. https://doi.org/10.3390/v11111057