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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
  • Fig. 1 Phosphoproteomic analysis of HipA-induced growth inhibition and HipB-induced resuscitation.

    (A) Growth curves of E. coli K-12 MG1655 carrying the pBAD33::hipA plasmid, in which hipA expression is under the control of an arabinose-inducible promoter, and the pNDM220::hipB plasmid, in which hipB is under the control of an IPTG-inducible promoter. Strains were grown in SILAC-labeled minimal medium containing stable isotope–labeled lysine derivatives: “light” lysine (Lys0), “medium-heavy” lysine (Lys4), or “heavy” lysine (Lys8), plus the appropriate antibiotics for retention of the plasmids. Expression of hipA was induced at OD600nm of 0.4 with arabinose, and samples were collected before (Lys0) and 75 min (Lys4) and 3 hours (Lys8) after induction. At the 3-hour time point, hipB expression was induced with IPTG, and samples were collected 2.5 hours (Lys4) and 6 hours (Lys8) later. Growth curves are representative of two independent experiments. (B and C) Distribution of phosphorylation site SILAC ratios 3 hours after hipA expression (B) and 6 hours after hipB expression (C). The names of the phosphorylated proteins and the positions of the phosphorylation sites showing at least a fourfold increase in phosphorylation (red) are indicated. Distributions are representative of two independent experiments. (D) Phosphorylation site profiles of GltX, RplK, and SeqA over four time points during growth inhibition (HipA 75 min and HipA 3 hours) and resuscitation (HipB 2.5 hours and HipB 6 hours) in two independent experiments. (E) Gene ontology (GO) distribution of those phosphoproteins showing at least a fourfold increase in phosphorylation 3 hours after hipA expression enriched against the background of all identified phosphoproteins (P < 0.01). The number of enriched phosphoproteins is indicated below the category name, and the names of the proteins involved in translation and DNA metabolism are provided. The distribution is representative of two independent experiments. (F) In vitro kinase assay of His6-HipA with His6-GltX, His6-RplK, and SeqA-His6. After the phosphorylation reaction, the samples were protease-treated and analyzed by LC-MS/MS. Increased phosphorylation at the indicated sites represented as circles was detected in two independent experiments. The smaller circle depicted for SeqA indicates a two-order-of-magnitude lower intensity of phosphorylation site without His6-HipA relative to the intensity measured in the presence of His6-HipA.

  • Fig. 2 Comparison of the phosphoproteomes of cells overproducing HipA or HipA7 in similar amounts.

    (A) Growth curves of MG1655 strains carrying either empty pBAD33 (pBAD), pBAD33::hipA (pBAD::hipA), or pBAD33::hipA7 (pBAD::hipA7), in which hipA or hipA7 expression is under the control of an arabinose-inducible promoter, and the pNDM220::hipB plasmid, in which hipB expression is under the control of an IPTG-inducible promoter. Strains were grown in SILAC-labeled minimal medium containing light lysine (Lys0), medium-heavy lysine (Lys4), or heavy lysine (Lys8) plus the appropriate antibiotics for retention of the plasmids. hipA or hipA7 expression was induced at OD600nm of 0.4 with arabinose, and samples were collected 95 min later. Expression of hipB was not induced in these experiments. Growth curves are representative of three independent experiments. (B and C) Distribution of phosphorylation site SILAC ratios 95 min after hipA7 expression relative to the empty plasmid (B) and relative to hipA expression (C). The names of the phosphorylated proteins and the positions of the phosphorylation sites showing at least a fourfold change in phosphorylation (red) are indicated. Distributions are representative of three independent experiments. (D) Occupancy of the HipA and HipA7 Ser150 autophosphorylation site (P site) after hipA or hipA7 expression determined by MaxQuant. (E) Occupancy of the SeqA Ser36 and (F) RplK Ser102 phosphorylation sites after hipA or hipA7 expression calculated manually. Data in (D) to (F) are means ± SD from three independent experiments.

  • Fig. 3 Phosphoproteomic analysis of cells producing low or high amounts of HipA or HipA7.

    (A) Experimental setup of two SILAC experiments in which hipA or hipA7 was induced from a low (pNDM220)– or a high (pMG25)–copy number plasmid, in which hipA or hipA7 is under the control of an IPTG-inducible promoter in MG1655 wt strain (for hipA expression) or hipBA deletion mutant (ΔhipBA, for hipA7 expression). Strains were grown in SILAC-labeled minimal medium containing light lysine (Lys0), medium-heavy lysine (Lys4), or heavy lysine (Lys8) plus ampicillin for retention of the plasmids. hipA or hipA7 expression was induced at OD600nm of 0.4 with IPTG, and samples were collected 95 min later. (B and C) Distribution of phosphorylation site SILAC ratios in cells, in which hipA (B) or hipA7 (C) was induced from the high–copy number plasmid relative to expression from the low–copy number plasmid. The names of the phosphorylated proteins and the positions of the phosphorylation sites showing at least a fourfold change in phosphorylation (red) are indicated. Distributions are representative of two independent experiments.

  • Fig. 4 Comparison of the phosphoproteomes of cells expressing hipA7 or hipA from the endogenous hipA chromosomal locus.

    (A) Growth curves of three MG1655 strains: a strain carrying the wild-type hipA gene (wt hipA), a deletion mutant lacking both hipB and hipAhipBA), and a strain carrying the hipA7 allele (hipA7) at the endogenous hipA locus. Strains were grown in SILAC-labeled minimal medium containing light lysine (Lys0), medium-heavy lysine (Lys4), or heavy lysine (Lys8), and samples were collected in the late stationary phase after 30 hours of growth. Growth curves are representative of three independent experiments. (B) Volcano plot of phosphorylation site SILAC ratios of the hipA7 strain relative to the wt hipA strain from three independent experiments. The black curve indicates statistical significance with the P value of 0.01 and a minimal fold change of 1. The names of phosphorylated proteins and the positions of the phosphorylation sites that statistically significantly increased or decreased in phosphorylation (red) are indicated. (C) Volcano plot of protein SILAC ratios of the hipA7 strain relative to the wt hipA strain from three independent experiments. The black curve indicates statistical significance with the P value of 0.001 and the minimal fold change of 1. Proteins statistically significantly increased or decreased in abundance are indicated in red. Names of HipB, PspA, and proteins of aromatic amino acid biosynthesis pathway are indicated in black text.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/547/eaat5750/DC1

    Fig. S1. Additional analysis of phosphoproteomic data from HipA-induced growth inhibition and HipB-induced resuscitation and follow-up experiments.

    Fig. S2. Additional analysis of phosphoproteomic data from comparably overproduced HipA and HipA7.

    Fig. S3. Additional analysis of phosphoproteomic data from low and high production of HipA and HipA7.

    Fig. S4. Additional analysis of phosphoproteomic data from hipA7 and wt hipA strains.

    Fig. S5. Functional analysis of the E. coli phosphoproteome obtained in this study.

    Table S1. Bacterial strains and plasmids.

    Table S2. DNA oligonucleotides.

    Table S3. Overview of all experiments measured by LC-MS/MS.

    Data file S1. Protein groups and phosphorylation sites identified in this study.

  • The PDF file includes:

    • Fig. S1. Additional analysis of phosphoproteomic data from HipA-induced growth inhibition and HipB-induced resuscitation and follow-up experiments.
    • Fig. S2. Additional analysis of phosphoproteomic data from comparably overproduced HipA and HipA7.
    • Fig. S3. Additional analysis of phosphoproteomic data from low and high production of HipA and HipA7.
    • Fig. S4. Additional analysis of phosphoproteomic data from hipA7 and wt hipA strains.
    • Fig. S5. Functional analysis of the E. coli phosphoproteome obtained in this study.
    • Table S1. Bacterial strains and plasmids.
    • Table S2. DNA oligonucleotides.
    • Table S3. Overview of all experiments measured by LC-MS/MS.
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Protein groups and phosphorylation sites identified in this study.

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