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

Ching-Chung Ko,Graham F Hatfull

doi:10.1038/s41598-020-71588-5

Abstract

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.

Highlights

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
Mycobacteriophage genomes have large numbers of genes of unknown function. Determining their roles in phage life cycles is complicated by the observation that many are not required for lytic growth[9]
They are often not conserved among groups of closely-related genomes, they frequently contribute to mosaic relationships and have relatives in unrelated g enomes[2]

Summary

Introduction

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. 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. The 3,300 sequenced actinobacteriophages include over 320,000 predicted protein coding genes, of which ~ 75% have no predicted function. Many of such genes are likely to be non-essential for viral lytic replication[9], most of them are expressed during lytic growth[10]. Some mycobacteriophage-encoded genes have been shown to be toxic including mycobacteriophage L5 genes 77, 78, and 7928,29, the product of one of which (gp77) interacts directly with the host protein M smeg_353230, and another (gp79) which promotes filamentation[28]

Methods

Results

Conclusion