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

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. The bacteriophage population is vast, dynamic, old, and highly diverse 1. Phage genome sizes vary considerably from 5 to 500 kbp, but the average size of referenced phage genome sequences in publicly available databases is ~ 50 kbp 2. The genome sequences of over 3,300 phages isolated on bacteria in the phylum Actinobacteria (i.e. 'actinobacteriophages') have been determined, with an average genome length of 61 kbp and an average of 100 predicted protein-coding genes per genome 3. With an average gene length of about 600 bp, phage genes are substantially smaller than the average bacterial gene, reflecting an abundance of relatively small genes coding for non-structural proteins 3,4. Phage genomes are characteristically mosaic as a consequence of horizontal genetic exchange 5,6 , although the rates of exchange vary for different types of phages 7. Mosaicism is likely driven largely by microbial dynamics involving bacterial resistance to infection and phage co-evolution 8. 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 , although most of them are expressed during lytic growth 10. The general roles for these proteins may be in modulating the dynamic interactions with their host, counteracting restriction, CRISPR-Cas, and other viral defense systems 11-13 , or to actively promote defense against heterotypic viral attack 11,14-16. Elucidating the roles and functions of these genes is of importance in understan...

Mycobacteriophages: From Petri dish to patient

“…It is likely that phage genes play important roles not just in host range determination, but in other aspects of viral dynamics, including out-competing other phages in lytic growth. Several studies have shown that a substantial proportion (approximately 20%) of phage genes are toxic to the host cell when expressed alone [ 82 , 83 ]. This toxicity itself is likely immaterial to lytic phage growth, as the cell is going to die anyway, and we suggest it more commonly derives from inactivation of cell processes that other phages depend on to lytically infect the bacterium.…”

“…An unrelated phage, Rosebush, requires DivIVA to infect, and is therefore excluded from Fruitloop-infected cells by this mechanism [ 82 ]. It seems likely that other genes identified as toxic are involved in similar processes [ 83 ].…”

Deconstructing the Phage–Bacterial Biofilm Interaction as a Basis to Establish New Antibiofilm Strategies

“…In this case, YIP was discovered by a growth inhibition screen [ 209 ]. Similarly, Ko and Hatfull (2020) screened 193 diverse mycobacteriophage genes from 13 different genomes and found that about 23% of them were toxic when expressed in Mycobacterium smegmatis [ 210 ]; this set of phage ORFans could easily be queried for biofilm impact.…”

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis

“…Other non-structural genes are tRNA and tmRNA, potentially involved in gene expression [ 80 , 83 ], mobile genetic elements (MGEs) [ 84 , 85 ], and several other genes of unknown function [ 55 ]. The latter do not seem to be required directly for phage replication, but instead might modulate bacteria-phage and/or phage-phage dynamic interactions [ 86 , 87 ]. Indeed, temperate mycobacteriophages, which can either undergo lytic or lysogenic cycle ( Figure 1 A), play important roles in these interactions both during lysogeny by prophage-mediated defense systems against the attack of other phages, and by superinfection exclusion during lytic replication, reviewed in detail elsewhere [ 77 , 88 , 89 , 90 ].…”