1. Molecular Mechanism of Bacterial Persistence by HipA
Elsa Germain, Daniel Castro-Roa, Nikolay Zenkin, Kenn Gerdes 
Molecular Cell 
Volume 52, Issue 2, Pages (October 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 52, 248-254DOI: (10.1016/j.molcel.2013.08.045)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
HipA Does Not Inhibit Translation by the Phosphorylation of EF-Tu
(A) Translation in vitro with an S30 extract without HipA (lanes 1–3) or with HipA (lanes 4–6). 0.1 μM HipA was incubated with the S30 extract for 10 min before the reaction was started by the addition of a DNA-template-encoding luciferase.
(B) A scheme of in vitro translation system reconstituted from purified components and stages 0.6 μM when HipA was added to the system: during the initiation step (blue), before formation of the ternary complex (EF-Tu⋅GTP⋅Phe-tRNAPhe) (purple), and to the preformed ternary complex (red). Bottom, the synthesis of Met-Phe dipeptide in the absence or presence of HipA added during the different stages explained above (lanes 3–5). As a control, the same experiment was performed by adding 0.2 μM EF-Tu kinase Doc (Castro-Roa et al., 2013); shown are no Doc added (lane 6) and Doc added before ternary complex formation (lane 7). Dipeptides were analyzed by thin-layer electrophoresis and autoradiography (Castro-Roa and Zenkin, 2012).
(C) Analysis of purified GST-EF-Tu (0.13 μM) after incubation for 45 min with (0.1 μM) HipA and 0.1 μM γ[32P]ATP by SDS-PAGE (left) and autoradiography (right). Only the autophosphorylation of HipA was observed (lanes 7–9) in comparison to the positive control with GST-EF-Tu phosphorylated by Doc kinase (lane 11). Experiments were reproduced at least three times.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2
GltX Counteracts HipA Toxicity, and HipA-Induced Persistence Depends on (p)ppGpp
(A) Strains MG1655ΔhipAB harboring pBAD30 (bla, vector plasmid carrying an arabinose-inducible promoter) or pEG3 (pBAD30::hipA) were transformed with pCA24N (cat, vector plasmid carrying an IPTG-inducible promoter) or pCA24N::gltX. The resulting four strains were plated on nutrient agar plates containing ampicillin (50 μg/ml), chloramphenicol (50 μg/ml), and arabinose (0.2%) without (left) or with (right) 50 μM IPTG, which induced gltX. As seen, the presence of the gltX-encoding plasmid suppressed HipA toxicity with and without IPTG (due to a slight leakiness of the IPTG-inducible promoter on the high-copy plasmid); as expected, the suppression of HipA toxicity was stronger with gltX induction (+IPTG).
(B) Growth curves of MG1655ΔhipBA containing the plasmids are indicated. Overnight cultures were diluted 1,000-fold in fresh rich medium with ampicillin (50 μg/ml), chloramphenicol (50 μg/ml), and 100 μM of IPTG (in order to induce gltX) and incubated at 37°C. The arrow indicates that hipA was induced at OD600 = 0.2 via the addition of 0.2% arabinose.
(C) Levels of hipA induced persistence in WT, ΔrelA, and Δ(relA spoT) strains. Exponentially growing cultures of MG1655 and its relA and relA spoT deletion derivatives containing pBAD30 control plasmid (−) or pEG4 (pBAD30::hipA) (+) were exposed to 2 μg/ml of ciprofloxacin (OD600 ≈0.5). The transcription of hipA was induced for 30 min before the addition of ciprofloxacin (t = 0). This panel shows the percentage of survival after 5 hr of antibiotic treatment (log scale). The bars show the averages of at least three independents experiments, and error bars indicate SD. The difference between ΔrelA and Δ(relA spoT) strains was not significant (Student’s t test; p = 0.2; n = 3).
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3
Phosphorylation of GltX In Vitro by HipA at Ser239 Requires tRNAGlu
(A) The phosphorylation of GltX in vitro. 6 μM GltX, 0.1 μM γ[32P]ATP, and 66 μM ATP were incubated with or without 0.2 μM HipA, 1.6 μM Glu, or 1.5 μM tRNAGlu for 45 min. In reactions where HipA was present, HipA was added first.
(B) Structures of the conserved KLSKR motif containing Ser239 phosphorylated by HipA. Shown are the ATP-bound form (green; PDB 1N75) and the ATP-Glu-tRNAGlu bound form (blue; PDB 1N77) of GltX. Rearrangement upon tRNAGlu binding is shown with a line. The numbering corresponds to amino acids of E. coli GltX.
(C) In vitro aminoacylation activity of GltXWT, GltX-P, and GltXS239D. Aminoacylation was tested in vitro in a reaction mixture containing 2 mM ATP, 0.6 μM GltX (WT, HipA treated or GltXS239D), 0.2 mg/ml tRNA, 100 μM glutamate cold, and [3H]-Glu (240 counts min−1 pmol−1) and, for experiments where GltX was phosphorylated, 0.6 μM HipA was used. The reaction was performed for 3 min at 37°C and quenched by precipitation in 5% TCA (Francklyn et al., 2008; Kern and Lapointe, 1981). Error bars indicate the SD of three independent experiments.
(D) GltXS239D was not phosphorylated by HipA in vitro. 6 μM GltX, 0.1 μM γ[32P]ATP, and 66 μM ATP were incubated with or without 0.2 μM HipA, 1.6 μM Glu, or 1.5 μM tRNAGlu for 45 min. In reactions where HipA was present, HipA was added first.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
Molecular Model Explaining HipA-Induced (p)ppGpp Synthesis and Persistence
(A) HipA is absent (or inactivated by HipB), glutamyl tRNA synthetase is active, and translation proceeds normally.
(B) HipA is active, GltX is inhibited by phosphorylation, and, therefore, uncharged tRNAGlu accumulates. Uncharged tRNAGlu loads at empty ribosomal A sites (“hungry” codons) that trigger the activation and release of RelA. The (p)ppGpp level increases and the stringent response is mounted. The model explains the highly pleiotropic cellular effects observed after hipA induction. HipA, pink; GltX, yellow; phosphate group, red; ribosome, gray. Charged and uncharged tRNAs are shown as sticks with or without filled circles, respectively. mRNA is shown as a wavy line. E, P, and A symbolize the ribosomal tRNA binding sites.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions