Department of Bacteriology and Immunology, Medicum, Human Microbiome Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland.
Division of Clinical Microbiology, HUSLAB, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, Finland.
Viruses. 2021 Apr 24;13(5):750. doi: 10.3390/v13050750.
Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an 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 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.
噬菌体能感染临床血液培养分离物，基于此能力，从污水样本中分离出的噬菌体 vB_EcoM_fHy-Eco03（简称 fHy-Eco03）。从 fHy-Eco03 的基因组序列中总共鉴定出 32 个编码未知功能的假定蛋白（HPUFs）的基因。利用一种新颖的基于下一代测序（NGS）的方法，对这些 HPUFs 进行毒性筛选（toxHPUFs）。该方法通过比较在电穿孔前的连接混合物的 DNA 中的基因特异性读覆盖率和电穿孔后的混合转化体，来鉴定 toxHPUF 编码基因。比较了 NGS 筛选测定法和基于平板效价的方法的性能和可靠性，两种方法均鉴定出 fHy-Eco03 基因产物具有毒性。虽然这两种筛选的结果高度相似，但 NGS 筛选测定法在可靠性和效率方面均优于平板效价测定法。NGS 筛选测定法可作为寻找新型噬菌体启发型抗菌分子的高通量方法。
