Background: 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.
Objectives: We postulated that the phenotypic difference between HipA and HipA7 may be driven by diverse substrate pools of the two kinase variants. We aimed to analyze regulatory networks involved in bacterial persistence using MS-based proteomics.
Methods: 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. We applied dynamic SILAC to study protein synthesis and turnover during HipA-mediated persistence and resuscitation.
Results: Our assays confirmed that both forms of the HipA kinase phosphorylate GltX as the main substrate. Importantly, HipA phosphorylated several additional substrates involved in translation, transcription and replication, such as ribosomal protein L11 and SeqA, which were further validated in vitro. 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. When expressed from the chromosome, HipA7 phosphorylated GltX and another substrate, a putative Ser/Thr kinase. Our preliminary data show that this novel kinase is also involved in persistence, revealing the first protein phosphorylation-based network involved in regulation of persistence. Our results contribute to understanding of HipA action and present a resource for future studies of bacterial persistence.