Relaxed Cleavage Specificity within the RelE Toxin Family

Abstract

Bacterial type II toxin-antitoxin systems are widespread in bacteria. Among them, the RelE toxin family is one of the most abundant. The RelE K-12 toxin of Escherichia coli K-12 represents the paradigm for this family and has been extensively studied, both in vivo and in vitro. RelE K-12 is an endoribonuclease that cleaves mRNAs that are translated by the ribosome machinery as these transcripts enter the A site. Earlier in vivo reports showed that RelE K-12 cleaves preferentially in the 5=-end coding region of the transcripts in a codon-independent manner. To investigate whether the molecular activity as well as the cleavage pattern are conserved within the members of this toxin family, RelE-like sequences were selected in Proteobacteria, Cyanobacteria, Actinobacteria, and Spirochaetes and tested in E. coli. Our results show that these RelE-like sequences are part of toxin-antitoxin gene pairs, and that they inhibit translation in E. coli by cleaving transcripts that are being translated. Primer extension analyses show that these toxins exhibit specific cleavage patterns in vivo, both in terms of frequency and location of cleavage sites. We did not observe codon-dependent cleavage but rather a trend to cleave upstream purines and between the second and third positions of codons, except for the actinobacterial toxin. Our results suggest that RelE-like toxins have evolved to rapidly and efficiently shut down translation in a large spectrum of bacterial species, which correlates with the observation that toxin-antitoxin systems are spreading by horizontal gene transfer.

Cell Growth Inhibition upon Deletion of Four Toxin-Antitoxin Loci from the Megaplasmids of Sinorhizobium meliloti

“…RelE toxicity results from cleavage of mRNA at the ribosomal A site as it is being translated (44). In a recent report, six RelE-like proteins from different phyla, including the 98-amino-acid SMc00822 protein from the chromosome of S. meliloti, were shown to inhibit translation by cleaving ribosome-associated transcripts (45). In general, cells exposed to the RelE toxin show a drastic growth arrest (39,46,47).…”

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

“…To avoid interference, the endoribonuclease activity of MazF from E. faecalis V583 was evaluated in the MG1655D10 E. coli strain, a K12 strain deleted for 10 TA systems encoding endoRNases, MG1655D10. 61 Total RNA was recovered after 0, 15, 30 and 60 minutes of induction of the toxin.…”

“…Parental E. coli K12 strains were DJ624Dara, 60 MG1655D10, 61 and DH5a (Invitrogen), and E. faecalis strains were V583, and OG1RF. 44,62 Plasmid pCRII-TOPO (Invitrogen) was used for direct cloning of amplicons.…”

Functional characterization of Aggregatibacter actinomycetemcomitans QSEBC : a bacterial adrengeric receptor and global regulator of virulence.

“…Based on the growth results in E. coli with the ectopic expression of D11S_2133 and D11S_2134, D11S_2133 is correctly annotated as a putative toxin and D11S_2134 is the anti-toxin, but in order to confirm these labels, functional assays will need to be performed. RelE toxins have been shown to act as endoribonucleases that cleave between the second and third position of an upstream purine [111]. In order to confirm that D11S_2133 acts as endoribonuclease, then different mRNA's with known sequences will be incubated together with the purified toxin and then ran on a gel to look for RNA degradation.…”

“…Toxin activity can vary but most toxins function as either ribosome dependent or independent mRNA endoribonucleases that cleave mRNA at certain specific sequences [81,109,110]. Some cleave mRNA promiscuously such as the RelE of E.coli which cleaves between the second and third position of an upstream purine [111]. TA systems are also thought to play a vital role in the development of the persister phenotype that allows the cell to survive environmental stress [81].…”