A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism

Regulation of the Escherichia coli HipBA Toxin-Antitoxin System by Proteolysis

Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon- background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence

The N-Terminus of GalE Induces tmRNA Activity in Escherichia coli

BACKGROUND: The tmRNA quality control system recognizes stalled translation complexes and facilitates ribosome recycling in a process termed 'ribosome rescue'. During ribosome rescue, nascent chains are tagged with the tmRNA-encoded SsrA peptide, which targets tagged proteins for degradation. In Escherichia coli, tmRNA rescues ribosomes arrested on truncated messages, as well as ribosomes that are paused during elongation and termination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a new translational pausing determinant that leads to SsrA peptide tagging of the E. coli GalE protein (UDP-galactose 4-epimerase). GalE chains are tagged at more than 150 sites, primarily within distinct clusters throughout the C-terminal domain. These tagging sites do not correspond to rare codon clusters and synonymous recoding of the galE gene had little effect on tagging. Moreover, tagging was largely unaffected by perturbations that either stabilize or destabilize the galE transcript. Examination of GalE-thioredoxin (TrxA) fusion proteins showed that the GalE C-terminal domain is no longer tagged when fused to an N-terminal TrxA domain. Conversely, the N-terminus of GalE induced tagging within the fused C-terminal TrxA domain. CONCLUSIONS/SIGNIFICANCE: These findings suggest that translation of the GalE N-terminus induces subsequent tagging of the C-terminal domain. We propose that co-translational maturation of the GalE N-terminal domain influences ribosome pausing and subsequent tmRNA activity

Relaxed cleavage specificity within the RelE toxin family.

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.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe