High persister protein kinase HipA and its gain-of-function variant HipA7 phosphorylate different substrate pools in Escherichia coli

ABSTRACT: Bacterial persistence, the ability to survive antibiotic treatment by entering a physiologically dormant state, is a serious biomedical problem. Protein Ser/Thr kinase HipA, the first toxin connected to bacterial multidrug tolerance (persistence), exerts its function by phosphorylating glutamate--tRNA ligase GltX, leading to a halt in translation, accumulation of ppGpp and induction of persistence. Intriguingly, its variant HipA7 is able to induce significantly higher levels of persistence despite being less toxic for the cell. We postulated that this phenotypic difference may be driven by diverse substrate pools of the two kinase variants. To address this, we ectopically expressed hipA and hipA7 in E. coli and monitored their in vivo substrates during growth inhibition and resuscitation using SILAC-based quantitative phosphoproteomics. Our assays confirmed that both forms of the kinase phosphorylate GltX as the main substrate. Importantly, HipA phosphorylated several additional substrates involved in protein synthesis, transcription and replication, such as ribosomal protein L11 and SeqA. Conversely, HipA7 had a lower kinase activity, no additional substrates under tested conditions and showed a similar substrate pool only when expressed at significantly higher levels. The two forms of the kinase also differed in autophosphorylation level, which was significantly lower in HipA7. Initial testing of the novel HipA substrate L11 did not reveal a connection between HipA-induced phosphorylation and RelA-dependent persistence, opening the possibility that the novel HipA substrates are predominantly responsible for the toxic phenotype of the kinase. Our results contribute to understanding of HipA7 action and present a resource for studies of HipA-related persistence.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Nicolas Nalpas  

LAB HEAD: Boris Macek

PROVIDER: PXD009173 | Pride | 2018-09-17

REPOSITORIES: Pride