Stine Vang Nielsen:
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Prokaryotic toxin-antitoxin systems are widespread and highly abundant genetic elements, typically consisting of two components: a toxin that inhibits an essential cellular process and an antitoxin that can counteract the toxin. The hipBA locus of Escherichia coli K-12 is a type II toxin-antitoxin system where hipA encodes a protein toxin that can be neutralized by the protein antitoxin encoded by hipB. HipA is a serine-threonine kinase that phosphorylates glutamyl-tRNA synthetase and thereby is capable of inhibiting cell growth and inducing the stringent response by increasing the production of the alarmone (p)ppGpp. hipA mutants presented the first genetic link to bacterial persistence, in which a bacterial subpopulation is antibiotic-tolerant. These mutants have also been found in disease-causing clinical E. coli isolates.
Here we present the discovery and characterization of a hipBA-homologous tripartite system encoded by the hipBST module of the pathogenic E. coli O127:H6 strain E2348/69. We show that HipT is a toxin capable of causing a transient and bacteriostatic growth inhibition, which can be counteracted by the HipS antitoxin, encompassing a Trp65 residue that is essential for its function. The third component, HipB, is a transcriptional repressor that additionally enhances the antitoxin functionality of HipS. We show that HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase (TrpS) at Ser197, which inhibits its aminoacylation activity. Consequently, this leads to an increase in uncharged tRNATrp, which induces the stringent response through (p)ppGpp accumulation. Furthermore, we determine that HipT has two autophosphorylation sites Ser57 and Ser59 and present structures of the HipBST complex in its native and two naturally occurring phosphorylated forms. We find that the two HipT autophosphorylation sites regulate its binding to HipS, and present evidence indicating that HipT in its phosphorylated forms is capable of phosphorylating TrpS. Finally, we show that the toxin-antitoxin complex is dynamic, which enables HipT to release and rebind HipS. Analysis of two additional hipBST-homologous systems from different bacterial species show that HipT and HipS have similar functionalities, whereas the effect of the third component as an antitoxin enhancer appears to have diverted. Taken together, we establish that hipBST is a novel family of three-component hipBA-related toxin-antitoxin systems.