Functional analysis of conserved hypothetical genes in the cluster K bacteriophage Hammy

Savannah Underwood, Amber Coats, Sara Dao, Grace Dittmar, J.C. Gardner, Taylor Gore, Taiya Jarva, Kathleen Johnson, Giorgi Kenkebashvili, Sudiksha Kumar, Jasmine Ransom, Gabriella Reyes, Chazmyn Riley, Daniel Sinclair, Breanna Smith, Audra Thompson, Garrett Watts, Victoria Williams, Clint Pablo, Danielle Heller, Viknesh Sivanathan, Dmitri Mavrodi

The genus Mycobacterium encompasses diverse saprophytic and commensal species, as well as serious pathogens such as M. tuberculosis and M. leprae. Bacteriophages play a crucial role in the evolution of mycobacteria and provide insights into the genetics and physiology of this economically important group of organisms. Mycobacteriophages also attract a lot of recent interest as potential therapeutic agents for the treatment of multidrug-resistant tuberculosis. To date, over 1,700 bacteriophages that infect Mycobacterium were characterized through genome sequencing and grouped into 29 clusters based on genetic similarity. In addition to well-characterized genes that encode structural, regulatory, DNA metabolism, and lytic proteins, all mycobacteriophages genomes carry numerous conserved hypothetical genes. The specific functions of these genes remain unknown, and only a few proteins have been expressed and studied experimentally.

In this study, we performed a functional analysis of Hammy, a K cluster mycobacteriophage with a 62-kb genome that encodes 95 predicted protein-coding genes. Fifty of these genes are homologous to viral proteins of known function, while the role of the remaining 45 genes is currently unknown. We employed a combination of high-fidelity PCR and Gibson assembly to clone 81 Hammy gene into the broad-host-range plasmid vector pSMEG-ExT (85% overall success rate). The resultant recombinant plasmids were electroporated into M. smegmatis mc² 155 and screened for cytotoxicity in the presence of the inducer anhydrotetracycline. The screen identified several cytotoxic genes, which are variably present in members of the K cluster (immediate relatives of Hammy) and other phages that infect Mycobacterium and Gordonia. Four cytotoxic genes (32, 34, 50, and 56) were subjected to bacterial two-hybrid analysis to identify the host proteins targeted by Hammy. The two-hybrid analysis identified several M. smegmatis proteins targeted by the cytotoxic gene 56. One of these targets, malate synthase, was previously identified during the two-hybrid analysis of bacteriophage ϕKMV and Pseudomonas aeruginosa. Results of this study will help to elucidate the role of poorly characterized viral genes in the biology of phages that infect M. smegmatis, M. tuberculosis, and closely related bacteria.