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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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An excess of pseudo-ToxI inhibits abortive infection.(A) Strains of Pba ToxIN (pTRB101) were tested for their ability to abort infection in the presence of a second, pBluescript II SK- based, antitoxic plasmid. Putatively antitoxic “test RNA” sequences were cloned under the control of the constitutive lacZα promoter, to allow for constant, high-level, expression. (B) EOPs of ΦTE, ΦM1 and ΦS61 on double strains of Pba, as per key, using Pba ToxIN-FS (pTRB102, pBluescript II SK-) as the control strain. Inserts in the second, antitoxic, plasmids are indicated by the horizontal axis labels. Plasmid pBluescript II SK- was used as the no insert, “vector”, control. “ΦTE escape locus” includes the escape locus from wild type ΦTE, whilst the “ΦTE genomic section” is a 269 bp region of the ΦTE genome, taken several kb from the escape locus as a negative control. Error bars indicate the standard deviation of triplicate (minimum) data.
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pgen-1003023-g006: An excess of pseudo-ToxI inhibits abortive infection.(A) Strains of Pba ToxIN (pTRB101) were tested for their ability to abort infection in the presence of a second, pBluescript II SK- based, antitoxic plasmid. Putatively antitoxic “test RNA” sequences were cloned under the control of the constitutive lacZα promoter, to allow for constant, high-level, expression. (B) EOPs of ΦTE, ΦM1 and ΦS61 on double strains of Pba, as per key, using Pba ToxIN-FS (pTRB102, pBluescript II SK-) as the control strain. Inserts in the second, antitoxic, plasmids are indicated by the horizontal axis labels. Plasmid pBluescript II SK- was used as the no insert, “vector”, control. “ΦTE escape locus” includes the escape locus from wild type ΦTE, whilst the “ΦTE genomic section” is a 269 bp region of the ΦTE genome, taken several kb from the escape locus as a negative control. Error bars indicate the standard deviation of triplicate (minimum) data.

Mentions: As the protection assays above (Figure 4D and 4E) relied on over-expression of the toxic and antitoxic components, it was considered that they may act as poor approximations to the relative stoichiometries of ToxN, ToxI and pseudo-ToxI during ΦTE infections. An assay was therefore designed to focus on whether Abi could be perturbed by excess antitoxin (Figure 6A). An excess was generated by first cloning both control and ΦTE-derived antitoxin sequences into the multiple cloning site of the high copy-number plasmid pBluescript II SK- (Fermentas), oriented so that they would be transcribed from the lacZα promoter. This constitutive promoter would ensure an over-abundance of our test RNA. These constructs were used in attempts to alter the Abi phenotype of an independent, pACYC184-based [26] ToxIN plasmid (pTRB101) within Pba.


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)

An excess of pseudo-ToxI inhibits abortive infection.(A) Strains of Pba ToxIN (pTRB101) were tested for their ability to abort infection in the presence of a second, pBluescript II SK- based, antitoxic plasmid. Putatively antitoxic “test RNA” sequences were cloned under the control of the constitutive lacZα promoter, to allow for constant, high-level, expression. (B) EOPs of ΦTE, ΦM1 and ΦS61 on double strains of Pba, as per key, using Pba ToxIN-FS (pTRB102, pBluescript II SK-) as the control strain. Inserts in the second, antitoxic, plasmids are indicated by the horizontal axis labels. Plasmid pBluescript II SK- was used as the no insert, “vector”, control. “ΦTE escape locus” includes the escape locus from wild type ΦTE, whilst the “ΦTE genomic section” is a 269 bp region of the ΦTE genome, taken several kb from the escape locus as a negative control. Error bars indicate the standard deviation of triplicate (minimum) data.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475682&req=5

pgen-1003023-g006: An excess of pseudo-ToxI inhibits abortive infection.(A) Strains of Pba ToxIN (pTRB101) were tested for their ability to abort infection in the presence of a second, pBluescript II SK- based, antitoxic plasmid. Putatively antitoxic “test RNA” sequences were cloned under the control of the constitutive lacZα promoter, to allow for constant, high-level, expression. (B) EOPs of ΦTE, ΦM1 and ΦS61 on double strains of Pba, as per key, using Pba ToxIN-FS (pTRB102, pBluescript II SK-) as the control strain. Inserts in the second, antitoxic, plasmids are indicated by the horizontal axis labels. Plasmid pBluescript II SK- was used as the no insert, “vector”, control. “ΦTE escape locus” includes the escape locus from wild type ΦTE, whilst the “ΦTE genomic section” is a 269 bp region of the ΦTE genome, taken several kb from the escape locus as a negative control. Error bars indicate the standard deviation of triplicate (minimum) data.
Mentions: As the protection assays above (Figure 4D and 4E) relied on over-expression of the toxic and antitoxic components, it was considered that they may act as poor approximations to the relative stoichiometries of ToxN, ToxI and pseudo-ToxI during ΦTE infections. An assay was therefore designed to focus on whether Abi could be perturbed by excess antitoxin (Figure 6A). An excess was generated by first cloning both control and ΦTE-derived antitoxin sequences into the multiple cloning site of the high copy-number plasmid pBluescript II SK- (Fermentas), oriented so that they would be transcribed from the lacZα promoter. This constitutive promoter would ensure an over-abundance of our test RNA. These constructs were used in attempts to alter the Abi phenotype of an independent, pACYC184-based [26] ToxIN plasmid (pTRB101) within Pba.

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