Research ResourceMicrobiology

The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance

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Science Signaling  11 Sep 2018:
Vol. 11, Issue 547, eaat5750
DOI: 10.1126/scisignal.aat5750

Balancing bacterial growth with drug resistance

Within some bacterial populations, a subset of cells grows more slowly than the rest, which decreases the competitive fitness of these cells under favorable growth conditions but enables them to survive exposure to antibiotics. The kinase HipA is important for the survival of such Escherichia coli persister cells because it targets the glutamate-tRNA ligase GltX, thus halting translation and slowing cell growth. A variant of this kinase that is associated with some clinical isolates, HipA7, is more efficient than HipA in inducing persistence, although it is less effective at reducing cell growth. Through phosphoproteomic analyses, Semanjski et al. found that although both HipA and HipA7 targeted GltX, HipA also targeted additional substrates, which likely account for the potency of HipA in reducing cell growth and may explain why HipA7, despite being more effective at promoting persistence, is less toxic than HipA.


The bacterial serine-threonine protein kinase HipA promotes multidrug tolerance by phosphorylating the glutamate-tRNA ligase (GltX), leading to a halt in translation, inhibition of growth, and induction of a physiologically dormant state (persistence). The HipA variant HipA7 substantially increases persistence despite being less efficient at inhibiting cell growth. We postulated that this phenotypic difference was caused by differences in the substrates targeted by both kinases. We overproduced HipA and HipA7 in Escherichia coli and identified their endogenous substrates by SILAC-based quantitative phosphoproteomics. We confirmed that GltX was the main substrate of both kinase variants and likely the primary determinant of persistence. When HipA and HipA7 were moderately overproduced from plasmids, HipA7 targeted only GltX, but HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation, SeqA. HipA7 showed reduced kinase activity compared to HipA and targeted a substrate pool similar to that of HipA only when produced from a high–copy number plasmid. The kinase variants also differed in autophosphorylation, which was substantially reduced for HipA7. When produced endogenously from the chromosome, HipA showed no activity because of inhibition by the antitoxin HipB, whereas HipA7 phosphorylated GltX and phage shock protein PspA. Initial testing did not reveal a connection between HipA-induced phosphorylation of RplK and persistence or growth inhibition, suggesting that other HipA-specific substrates were likely responsible for growth inhibition. Our results contribute to the understanding of HipA7 action and present a resource for elucidating HipA-related persistence.

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