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Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

Blower TR, Evans TJ, Przybilski R, Fineran PC, Salmond GP - PLoS Genet. (2012)

Bottom Line: The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination.This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system.Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

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Related in: MedlinePlus

Analysis of pseudo-ToxI as a potential antitoxin.(A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.
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pgen-1003023-g004: Analysis of pseudo-ToxI as a potential antitoxin.(A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.

Mentions: An alignment of the ToxI RNA sequence with the predicted pseudo-ToxI RNA showed that the majority of nucleotide positions were conserved (Figure 4A). When the mutated positions were mapped onto the structure of antitoxic ToxI RNA in complex with the ToxN protein [13], they formed five groups as defined by their proximity within the structure (Figure 4B and 4C). To ascertain whether the aligned pseudo-ToxI was in fact a functional antitoxin, a single repeat was cloned into an expression vector, pTA100 [11] and tested for the ability to inhibit ToxN over-expressed from a second, pBAD30-based [25] vector, within an E. coli model (Figure 4D). In this assay, pseudo-ToxI could not suppress ToxN toxicity (Figure 4D). Each mutation group was therefore examined in isolation to compare the contribution each made to rendering pseudo-ToxI inactive within this assay (Figure 4E). Groups 1, 2, 3 and 5 had no impact on the antitoxic activity of ToxI, alone or in concert, whilst group 4 was solely responsible for knocking-out ToxI function (Figure 4E). Group 4 comprised three contiguous nucleotides. The contribution of each individual group 4 nucleotide substitution was assessed. Mutations C27G and U28A were each independently sufficient to knock-out ToxI, either in the pseudo-ToxI (ie. with mutations 1, 2, 3 and 5) or ToxI backgrounds (Figure 4E). As pseudo-ToxI was not active as a single, aligned, repeat (Figure 4D), it was also decided to clone and test the whole escape loci from ΦTE wt, ΦTE-A and ΦTE-F (Figure 4F). Only the sequence from ΦTE-F (which had recombined with ToxI) was antitoxic (Figure 4F).


Viral evasion of a bacterial suicide system by RNA-based molecular mimicry enables infectious altruism.

Blower TR, Evans TJ, Przybilski R, Fineran PC, Salmond GP - PLoS Genet. (2012)

Analysis of pseudo-ToxI as a potential antitoxin.(A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003023-g004: Analysis of pseudo-ToxI as a potential antitoxin.(A) Alignment of the pseudo-ToxI and ToxI RNA sequences. Pseudo-ToxI nucleotides are coloured to match (B) and (C), with the green and purple bases denoting the 5′ and 3′ ends of a single pseudoknot, respectively. Mutated nucleotides in pseudo-ToxI are coloured orange and numbered according to their grouping, whilst the asterisk indicates the additional 3′ nucleotide. The dotted line connecting the U in group 3 indicates the uracil that is deleted in the case of expanded repeats with 2T sequences rather than 3T. (B) Schematic of the ToxI pseudoknot. Each position containing a mutation in the pseudo-ToxI RNA has been bracketed, with the ToxI base separated from the pseudo-ToxI base by a ‘/’. The mutations have been grouped 1–5, according to position, and highlighted in orange, with the 5′ and 3′ termini in green and violet, respectively. Indels, such as U17 that is deleted in some pseudo-ToxI repeats, and the additional A* inserted in all, have been bordered by a dashed line. Base interactions are indicated by black lines, and duplex and triplex base-interactions are bordered in grey. (C) Detail of the ToxIN trimer with each pseudoknot shown either in blue, purple or beige. Each ToxN monomer is shown as a grey surface. The blue pseudoknot is oriented relative to (B). The positions of mutation groups are shown, with the group number encircled in the same colour as the corresponding pseudoknot. The additional nucleotide of group 5 is not visible as this was not in the original solved ToxIN structure. PDB: 2XDB. (D) Pseudo-ToxI cannot protect from ToxN in an over-expression assay. Protection assays were conducted as per Materials and Methods using strains of E. coli DH5α carrying both pTA49 (inducible ToxN) and a second inducible antitoxin vector as shown, including use of pTA100 as a vector-only control, “vector”. Error bars indicate the standard deviation of triplicate data. (E) Protection assays using mutants of ToxI carried out as in (D) with the antitoxin mutations in each construct numbered as per (B). (F) Protection assays carried out as in (D), testing the full escape loci of ΦTE wt, ΦTE-A and ΦTE-F with full ToxI as a positive control. Under these conditions, there was sufficient antitoxin present to inhibit induced ToxN even without specific induction of the ToxI and ΦTE-F constructs.
Mentions: An alignment of the ToxI RNA sequence with the predicted pseudo-ToxI RNA showed that the majority of nucleotide positions were conserved (Figure 4A). When the mutated positions were mapped onto the structure of antitoxic ToxI RNA in complex with the ToxN protein [13], they formed five groups as defined by their proximity within the structure (Figure 4B and 4C). To ascertain whether the aligned pseudo-ToxI was in fact a functional antitoxin, a single repeat was cloned into an expression vector, pTA100 [11] and tested for the ability to inhibit ToxN over-expressed from a second, pBAD30-based [25] vector, within an E. coli model (Figure 4D). In this assay, pseudo-ToxI could not suppress ToxN toxicity (Figure 4D). Each mutation group was therefore examined in isolation to compare the contribution each made to rendering pseudo-ToxI inactive within this assay (Figure 4E). Groups 1, 2, 3 and 5 had no impact on the antitoxic activity of ToxI, alone or in concert, whilst group 4 was solely responsible for knocking-out ToxI function (Figure 4E). Group 4 comprised three contiguous nucleotides. The contribution of each individual group 4 nucleotide substitution was assessed. Mutations C27G and U28A were each independently sufficient to knock-out ToxI, either in the pseudo-ToxI (ie. with mutations 1, 2, 3 and 5) or ToxI backgrounds (Figure 4E). As pseudo-ToxI was not active as a single, aligned, repeat (Figure 4D), it was also decided to clone and test the whole escape loci from ΦTE wt, ΦTE-A and ΦTE-F (Figure 4F). Only the sequence from ΦTE-F (which had recombined with ToxI) was antitoxic (Figure 4F).

Bottom Line: The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination.This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system.Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Abortive infection, during which an infected bacterial cell commits altruistic suicide to destroy the replicating bacteriophage and protect the clonal population, can be mediated by toxin-antitoxin systems such as the Type III protein-RNA toxin-antitoxin system, ToxIN. A flagellum-dependent bacteriophage of the Myoviridae, ΦTE, evolved rare mutants that "escaped" ToxIN-mediated abortive infection within Pectobacterium atrosepticum. Wild-type ΦTE encoded a short sequence similar to the repetitive nucleotide sequence of the RNA antitoxin, ToxI, from ToxIN. The ΦTE escape mutants had expanded the number of these "pseudo-ToxI" genetic repeats and, in one case, an escape phage had "hijacked" ToxI from the plasmid-borne toxIN locus, through recombination. Expression of the pseudo-ToxI repeats during ΦTE infection allowed the phage to replicate, unaffected by ToxIN, through RNA-based molecular mimicry. This is the first example of a non-coding RNA encoded by a phage that evolves by selective expansion and recombination to enable viral suppression of a defensive bacterial suicide system. Furthermore, the ΦTE escape phages had evolved enhanced capacity to transduce replicons expressing ToxIN, demonstrating virus-mediated horizontal transfer of genetic altruism.

Show MeSH
Related in: MedlinePlus