Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay
Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an <i>Escherichia coli</i> clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genom...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-04-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/13/5/750 |
| _version_ | 1827694238220943360 |
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| author | Jutta Kasurinen Cindy M. Spruit Anu Wicklund Maria I. Pajunen Mikael Skurnik |
| author_facet | Jutta Kasurinen Cindy M. Spruit Anu Wicklund Maria I. Pajunen Mikael Skurnik |
| author_sort | Jutta Kasurinen |
| collection | DOAJ |
| description | Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an <i>Escherichia coli</i> clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene <i>g05</i> product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules. |
| first_indexed | 2024-03-10T12:00:20Z |
| format | Article |
| id | doaj.art-dc7ea16c2ea54159ba32b0cc07f48993 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T12:00:20Z |
| publishDate | 2021-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-dc7ea16c2ea54159ba32b0cc07f489932023-11-21T17:01:45ZengMDPI AGViruses1999-49152021-04-0113575010.3390/v13050750Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based AssayJutta Kasurinen0Cindy M. Spruit1Anu Wicklund2Maria I. Pajunen3Mikael Skurnik4Department of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, FinlandDepartment of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, FinlandDepartment of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, FinlandDepartment of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, FinlandDepartment of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, FinlandBacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an <i>Escherichia coli</i> clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene <i>g05</i> product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.https://www.mdpi.com/1999-4915/13/5/750bacteriophagehypothetical proteins of unknown functionnext-generation sequencingtoxic protein screen |
| spellingShingle | Jutta Kasurinen Cindy M. Spruit Anu Wicklund Maria I. Pajunen Mikael Skurnik Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay Viruses bacteriophage hypothetical proteins of unknown function next-generation sequencing toxic protein screen |
| title | Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay |
| title_full | Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay |
| title_fullStr | Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay |
| title_full_unstemmed | Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay |
| title_short | Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay |
| title_sort | screening of bacteriophage encoded toxic proteins with a next generation sequencing based assay |
| topic | bacteriophage hypothetical proteins of unknown function next-generation sequencing toxic protein screen |
| url | https://www.mdpi.com/1999-4915/13/5/750 |
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