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Functional Characterization of Bacteriophage Gene Product Toxic to Mycobacterium Smegmatis

AJAS · 2025 Microbiology (inferred)

Ava Blackledge · Kentucky Academy of Science

Overview

Antibiotic resistance is a critical global health challenge, with the UN estimating that drug-resistant infection deaths will surpass cancer deaths by 2050. Bacteriophages offer a potential alternative to traditional antibiotics, which are becoming increasingly ineffective due to resistance. MooMoo is a mycobacteriophage that is genetically distinct from all known phages (exhibiting <35% DNA similarity with known phages). Bioinformatic analysis identified several MooMoo genes, including MooMoo gene 51 (MM51), as having no known function. MM51 was later shown to be extremely toxic to its host, M. smegmatis. Using a bacterial two-hybrid assay, this project aimed to characterize MM51 by identifying interactions between the phage and host proteins, revealing mechanisms of MM51’s toxicity. Identifying the host-interacting partner could lead to the development of a new therapeutic. The MM51 gene sequence was previously cloned into the pExTra shuttle vector via isothermal assembly. The plasmid was moved into M. smeg cells to assess cytotoxicity, and MM51 was shown to prevent growth when overproduced. After confirming the protein's toxicity to host cells, the gene was subcloned into the p2Hα two-hybrid vector and transformed into chemically competent E. coli cells. After clone verification, the two-hybrid assay was performed. A positive interaction was detected by blue colony growth in the presence of the antibiotic carbenicillin. The clone that produced a positive interaction was sequenced to identify the host protein. Six out of sixteen clones produced positive interactions, but each clone was identified as a different essential host gene fragment. These results indicated MM51 has multiple interacting partners.

Competition history

  • AJAS 2025 Category not listed

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Source: AAAS Annual Meeting (Confex) / American Junior Academy of Science

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