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

Abstract

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 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.

hipBA toxin-antitoxin systems mediate persistence in Caulobacter crescentus

“…E. coli HipA phosphorylates GltX on Ser 239 within a conserved motif that binds and positions the catalytic ATP molecule 25,26 . Phosphorylation by E. coli HipA or substitution of an aspartate residue for Ser 239 blocked the tRNA charging activity of GltX in vitro 25 .…”

“…(p)ppGpp binding to target proteins, such as RNA polymerase and primase, reprograms transcription, downregulates macromolecular syntheses, and promotes dormancy 28 . However, other substrates of E. coli HipA have recently been identified, and additional phosphorylation events may stimulate persister formation 26 .hipBA operons are present in numerous, phylogenetically distinct bacterial genomes, yet it is unknown if all HipBA modules influence persistence, or if all HipA toxins phosphorylate the same substrates. To address these questions, we are studying HipBA systems in Caulobacter crescentus NA1000, a Gram-negative alpha-proteobacterium that lives in nutrient-limited freshwater environments and maintains three hipBA operons in its chromosome 29,30 .…”

“…(p)ppGpp binding to target proteins, such as RNA polymerase and primase, reprograms transcription, downregulates macromolecular syntheses, and promotes dormancy 28 . However, other substrates of E. coli HipA have recently been identified, and additional phosphorylation events may stimulate persister formation 26 .…”

An interaction network of RNA-binding proteins involved inDrosophilaoogenesis

“…Peptides were desalted Interactome of RNA Binding Proteins from Drosophila ovaries 8 and purified on C18 StageTips (68). LC-MS analysis were performed on a nanoLC (Easy-nLC 1200, Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) through a nanoelectrospray ion source (Thermo Fisher Scientific), as described previously (69). In brief, peptides were eluted using a segmented gradient of 10%-50% HPLC solvent B (80% ACN in 0.1% formic acid) at a flow rate of 200 nL/min over 46 min.…”

Importance of protein Ser/Thr/Tyr phosphorylation for bacterial pathogenesis

“…Moreover, species-specific optimization may be required to maximize protocol efficiencies [139]. The recent acquisition of large bacterial phosphoproteomes has allowed the assessment of phosphosite sequence features, representing putative kinase Phosphorylation consensus sequences have been identified in datasets obtained from M. tuberculosis [51,125], Helicobacter pylori [145], S. flexneri [64], and E. coli [63,146,147], including bacteria-specific N-and C-terminal phosphorylation preferences [139,147]. Interestingly, a phosphoproteomic dataset from the tick-transmitted Ehrlichia ruminantium pathogen was enriched in recognition motifs for several eukaryal kinases, suggesting significant host cell interaction [148].…”