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Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

ABSTRACT: Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous "escape" mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5-10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa.

SUBMITTER: Chen B

PROVIDER: S-EPMC5449768 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

Chen Bihe B Akusobi Chidiebere C Fang Xinzhe X Salmond George P C GPC

Frontiers in microbiology 20170531

Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxIN<sub>Pa</sub> consisting of an endoribonuclease toxin and RNA antitoxin. ToxIN<sub>Pa</sub> is a heterohexameric quaternary complex in which ...[more]

PMID: 28620370

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Project description:BackgroundBacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank.ResultsHere, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs) that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases.ConclusionOur observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4-like phages harbour a wealth of genetic material that has not been identified previously. The mechanisms by which these genes may have arisen may differ from those previously proposed for the evolution of other bacteriophage genomes.

Project description:Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection [Abi]). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage ΦM1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ΦM1 is a member of the Podoviridae and a member of the "KMV-like" viruses, a subset of the T7 supergroup. Genomic sequencing of ΦM1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when overexpressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was overexpressed, it copurified with the host nucleotide excision repair protein UvrA. While the M1-23 protein interacted with UvrA in coimmunoprecipitation assays, a UvrA mutant strain still aborted ΦM1, suggesting that the interaction is not critical for the type III TA Abi activity. Additionally, ΦM1 escaped a heterologous type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown, but further analysis of this protein may provide insights into the mode of activation of both systems.IMPORTANCE Bacteriophages, the viral predators of bacteria, are the most abundant biological entities and are important factors in driving bacterial evolution. In order to survive infection by these viruses, bacteria have evolved numerous antiphage mechanisms. Many of the studies involved in understanding these interactions have led to the discovery of biotechnological and gene-editing tools, most notably restriction enzymes and more recently the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Abortive infection is another such antiphage mechanism that warrants further investigation. It is unique in that activation of the system leads to the premature death of the infected cells. As bacteria infected with the virus are destined to die, undergoing precocious suicide prevents the release of progeny phage and protects the rest of the bacterial population. This altruistic suicide can be caused by type III toxin-antitoxin systems, and understanding the activation mechanisms involved will provide deeper insight into the abortive infection process.

Project description:The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

Dataset Information

Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

Publications

Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

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