Identification of mycobacteriophage toxic genes reveals new features of mycobacterial physiology and morphology

Double-stranded DNA tailed bacteriophages typically code for 50–200 genes, of which 15–35 are involved in virion structure and assembly, DNA packaging, lysis, and DNA metabolism. However, vast numbers of other phage genes are small, are not required for lytic growth, and are of unknown function. The 1,885 sequenced mycobacteriophages encompass over 200,000 genes in 7,300 distinct protein ‘phamilies’, 77% of which are of unknown function. Gene toxicity provides potential insights into function, and here we screened 193 unrelated genes encoded by 13 different mycobacteriophages for their ability to impair the growth of Mycobacterium smegmatis . We identified 45 (23%) mycobacteriophage genes that are toxic when expressed. The impacts on M. smegmatis growth range from mild to severe, but many cause irreversible loss of viability. Expression of most of the severely toxic genes confers altered cellular morphologies, including filamentation, polar bulging, curving, and, surprisingly, loss of viability of one daughter cell at division, suggesting specific impairments of mycobacterial growth. Co-immunoprecipitation and mass spectrometry show that toxicity is frequently associated with interaction with host proteins and alteration or inactivation of their function. Mycobacteriophages thus present a massive reservoir of genes for identifying mycobacterial essential functions, identifying potential drug targets and for exploring mycobacteriophage physiology.