Serine-Threonine Kinases Encoded by Split hipA Homologs Inhibit Tryptophanyl-tRNA Synthetase

Type II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact. hipBA of Escherichia coli strain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of hipA inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed that trpS in high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT of E. coli O127 is a kinase that phosphorylates tryptophanyl-tRNA synthetase in vitro at a conserved serine residue. Consistently, induction of hipT inhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream from hipT, called hipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipS in vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additional hipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus, hipBST constitutes a novel family of tricomponent TA modules where hipA has been split into two genes, hipS and hipT, that function as a novel type of TA pair.