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Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.

Blower TR, Short FL, Rao F, Mizuguchi K, Pei XY, Fineran PC, Luisi BF, Salmond GP - Nucleic Acids Res. (2012)

Bottom Line: The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria.Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages.This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.

ABSTRACT
Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

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Related in: MedlinePlus

Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously (9); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli, which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.
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gks231-F5: Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously (9); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli, which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.

Mentions: To determine whether the putative systems function as Type III TA loci, we assessed the toxicity of the proposed toxin gene and the ability of the cognate antitoxic repeats to inhibit the lethal effects. The toxin genes from the tenpIN family P. luminescens TT01 locus, and cptIN family C. catus GD/7, R. torques L2-14 and E. rectale DSM 17629 loci were cloned under the control of the l-arabinose inducible promoter in pBAD30 (33), including the native ribosome binding site. The respective antitoxic repeats, either as single repeats or as the full tandem array, were cloned into a spectinomycin-resistant derivative of pQE-80L, pTA100 (9), where they could be over-expressed by addition of IPTG. By co-transforming E. coli DH5α with both toxin and antitoxin plasmids, we were able to assess the toxicity and antitoxicity of each component (Figure 5). In the case of P. luminescens, a single TenpI repeat did not provide antitoxicity, over and above the empty vector control (Figure 5). However, the full TenpI locus of P. luminescens, acted to inhibit the cognate toxin (Figure 5). This implies that the active pseudoknot from this locus is encoded over the DNA repeat boundary, so at least two DNA repeats are required to form at least one active inhibitory pseudoknot. Following the results from P. luminescens, the full antitoxins were cloned from C. catus, R. torques and E. rectale. These loci acted as expected, with the toxin inhibited by the cognate antitoxic RNA. The cloned antitoxin sequences do not encode any predicted, translatable, open reading frames, supporting the model that the repeats encode antitoxic pseudoknots of RNA, rather than any antitoxic peptide.Figure 5.


Identification and classification of bacterial Type III toxin-antitoxin systems encoded in chromosomal and plasmid genomes.

Blower TR, Short FL, Rao F, Mizuguchi K, Pei XY, Fineran PC, Luisi BF, Salmond GP - Nucleic Acids Res. (2012)

Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously (9); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli, which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401426&req=5

gks231-F5: Protection of E. coli DH5α from Type III toxins by cognate antitoxins. Protection assays were performed as described in Materials and Methods. Results for the toxIN system of P. atrosepticum have been published previously (9); data from a single toxIN experiment is included for illustrative purposes. Of the four new loci tested, all toxin genes reduced viability of the host E. coli, which could then be restored by the full cognate antitoxin. Data shown are the mean values from triplicate experiments, with standard deviations represented by error bars.
Mentions: To determine whether the putative systems function as Type III TA loci, we assessed the toxicity of the proposed toxin gene and the ability of the cognate antitoxic repeats to inhibit the lethal effects. The toxin genes from the tenpIN family P. luminescens TT01 locus, and cptIN family C. catus GD/7, R. torques L2-14 and E. rectale DSM 17629 loci were cloned under the control of the l-arabinose inducible promoter in pBAD30 (33), including the native ribosome binding site. The respective antitoxic repeats, either as single repeats or as the full tandem array, were cloned into a spectinomycin-resistant derivative of pQE-80L, pTA100 (9), where they could be over-expressed by addition of IPTG. By co-transforming E. coli DH5α with both toxin and antitoxin plasmids, we were able to assess the toxicity and antitoxicity of each component (Figure 5). In the case of P. luminescens, a single TenpI repeat did not provide antitoxicity, over and above the empty vector control (Figure 5). However, the full TenpI locus of P. luminescens, acted to inhibit the cognate toxin (Figure 5). This implies that the active pseudoknot from this locus is encoded over the DNA repeat boundary, so at least two DNA repeats are required to form at least one active inhibitory pseudoknot. Following the results from P. luminescens, the full antitoxins were cloned from C. catus, R. torques and E. rectale. These loci acted as expected, with the toxin inhibited by the cognate antitoxic RNA. The cloned antitoxin sequences do not encode any predicted, translatable, open reading frames, supporting the model that the repeats encode antitoxic pseudoknots of RNA, rather than any antitoxic peptide.Figure 5.

Bottom Line: The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria.Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages.This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.

ABSTRACT
Toxin-antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin-antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin-antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin-antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin-antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

Show MeSH
Related in: MedlinePlus