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Molecular Mechanism of Bacterial Persistence by HipA
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell
Short Article Molecular Mechanism of Bacterial Persistence by HipA Elsa Germain,1,2 Daniel Castro-Roa,1,2 Nikolay Zenkin,1,* and Kenn Gerdes1,* 1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, NE2 4AX Newcastle upon Tyne, UK 2These authors contributed equally to this work *Correspondence: [email protected] (N.Z.), [email protected] (K.G.) http://dx.doi.org/10.1016/j.molcel.2013.08.045
SUMMARY
HipA of Escherichia coli is a eukaryote-like serinethreonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser239 near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNAGlu-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser239 changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of ‘‘hungry’’ codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.
INTRODUCTION Bacteria can enter a physiological state, called persistence or multidrug tolerance, in which lethal antibiotics do not kill them (Bigger, 1944). Persistence is a phenotype expressed by almost all bacteria, including major pathogens, and is believed to contribute to the intractability of chronic and relapsing infections (Levin and Rozen, 2006; Lewis, 2010). Experiments employing E. coli as the model organism indicated that persister cells form stochastically by switching into and out of slow growth (Balaban et al., 2004), a phenotype that was argued to be advantageous in changing environments (Kussell et al., 2005). Importantly, descendants of persister cells are as sensitive as their ancestors toward the bactericidal antibiotic used, demonstrating that bacterial persistence is a noninherited epigenetic characteristic (Lewis, 2010). The isolation of high-persistence (hip) mutants in E. coli indicated that persistence can have a genetic basis (Moyed and Ber-
trand, 1983). E. coli cells carrying hipA7, the most thoroughly analyzed hip mutant, exhibited a dramatic 100- to 1,000-fold increase in persistence (Korch et al., 2003). A low level of induction of hipA induced a bacteriostatic condition that could be counteracted by HipB encoded by the gene just upstream to hipA (Korch and Hill, 2006). Moreover, HipA and HipB formed a tight complex that autoregulated the transcription of the hipBA operon (Black et al., 1994). Due to these observations, it was suggested that hipBA constitutes a type II toxin-antitoxin (TA) locus (Korch et al., 2003), a suggestion that was later confirmed experimentally (Korch and Hill, 2006; Schumacher et al., 2009). Most TA loci encode two components, a toxin whose ectopic production inhibits cell growth and an antitoxin (RNA or protein) that counteracts toxin expression or activity (Blower et al., 2011; Gerdes and Maisonneuve, 2012). In type II TA loci, which are almost ubiquitous in bacteria and archaea, the antitoxins are proteins that interact with and neutralize the activity of the toxins (Gerdes et al., 2005). Most type II TA loci encode inhibitors of translation whose ectopic expression induces a static, drugtolerant condition from which the cells can be resuscitated by the induction of the antitoxin-encoding gene, consistent with a role in persistence (Correia et al., 2006; Han et al., 2010; Maisonneuve et al., 2011; Pedersen et al., 2002; Singh et al., 2010; Va´zquez-Laslop et al., 2006). E. coli has 11 canonical type II TA loci, ten of which encode toxins that inhibit translation by mRNA cleavage (Gerdes and Maisonneuve, 2012). Many type II TA operon mRNAs were significantly increased in persister cells (Keren et al., 2004; Shah et al., 2006), and the progressive deletion of these TA loci led to a gradual reduction in persistence, convincingly arguing that there is a causal connection between type II TA loci and persistence (Maisonneuve et al., 2011). Because hipA of E. coli was the first ‘‘persister’’ gene to be discovered, it has been of considerable interest in understanding how HipA inhibits cell growth and confers persistence. The ectopic induction of hipA induced growth arrest and strongly inhibited replication, transcription, and translation (Korch and Hill, 2006). Interestingly, HipA exhibits a eukaryotic serine-threonine kinase-like fold and has kinase activity (Correia et al., 2006; Schumacher et al., 2009). On the basis of in vitro experiments, it was suggested that HipA inhibits translation by the phosphorylation of EF-Tu (Schumacher et al., 2009). However, such phosphorylation did not explain the strong inhibition of replication and transcription seen after the induction of hipA (Korch and Hill, 2006; Schumacher et al., 2009). Moreover, hipA induction Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 1
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
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 mM 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 mM when HipA was added to the system: during the initiation step (blue), before formation of the ternary complex (EFTu,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 mM EF-Tu kinase Doc (CastroRoa 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 mM) after incubation for 45 min with (0.1 mM) HipA and 0.1 mM g[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.
stimulated RelA-dependent synthesis of (p)ppGpp (Bokinsky et al., 2013), thus raising the possibility that HipA has additional cellular targets. Here, we re-examine the molecular mechanism underlying HipA-induced persistence and show that the target of HipA is glutamyl-tRNA synthetase (GltX), which is inactivated by phosphorylation by HipA. Our results explain previous enigmatic physiological effects seen after the induction of hipA. RESULTS HipA Does Not Inhibit Translation by the Phosphorylation of EF-Tu HipA inactivates itself by autophosphorylation (Correia et al., 2006), which does not occur in the presence of HipB (Evdokimov et al., 2009). Therefore, we purified HipA in complex with HipB, as described previously (Christensen-Dalsgaard et al., 2008). After purification, MALDI-TOF mass spectrometry analysis revealed that more than 65% of the HipA molecules were nonphosphorylated (Figure S1A). We tested whether HipA could affect translation in vitro. As seen in Figure 1A, HipA efficiently inhibited the synthesis of luciferase in a cell-free translation system on the basis of a crude S30 cell extract. To identify the natural target of HipA, we used an in vitro translation system assembled from purified components (Castro-Roa and Zenkin, 2012). The mRNA used in this system encoded the dipeptide Met-Phe (MF) in its initial sequence. Translation was initiated by the addition of purified ribosomes, initiation factors IF-1, IF-2, IF-3, and [35S]-fMet-tRNAfMet. Then, initiated ribosomes were allowed to elongate by one codon via the addition of preformed ternary complex EF-Tu:GTP:PhetRNAPhe and the elongation factor EF-G in the presence of GTP. The short peptide products of the reaction were analyzed by thin layer electrophoresis (Castro-Roa and Zenkin, 2012; Zaher and Green, 2009). HipA was added to the reactions at three different stages: (1) during initiation complex formation (blue in Figure 1B), (2) before ternary complex formation (purple in Figure 1B), and (3) to the preformed ternary complex (TC) 2 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
(red in Figure 1B). As a positive control, we used Doc, a kinase that inhibits translation by the phosphorylation of EF-Tu (Castro-Roa et al., 2013) (Figure 1B). Surprisingly, although Doc efficiently inhibited the formation of the MF dipeptide (Figure 1B, lane 7), HipA had no effect on the reaction in any of the setups (Figure 1B, lanes 3–5). The above result contradicts the previously proposed model in which HipA inactivates translation by the phosphorylation of EFTu (Schumacher et al., 2009). Furthermore, to test this earlier model, we analyzed the phosphorylation of EF-Tu by HipA using g[32P]ATP. Given that HipA and EF-Tu migrate similarly in SDSPAGE, we used a functional GST-tagged version of EF-Tu to improve separation (Perla-Kajan et al., 2010). As seen from Figure 1C, no phosphorylation of GST-EF-Tu by HipA was observed (Figure 1C, lane 9). In contrast, GST-EF-Tu was efficiently phosphorylated by the Doc kinase (Castro-Roa et al., 2013) (Figure 1C, lane 11). Next, we decided to follow the phosphorylation state of EF-Tu in vivo. We simultaneously overproduced HipA and EF-Tu (in a strain lacking hipBA in order to avoid the neutralization of HipA by the endogenous antitoxin HipB). Then, EF-Tu purified before or after HipA overproduction was analyzed by mass spectrometry. As seen from Figure S1B (available online), we did not detect phosphorylation of EF-Tu. Altogether, our results show that HipA does not phosphorylate EF-Tu and that EF-Tu is not the target of HipA during the inactivation of translation.
Figure 2. GltX Counteracts HipA Toxicity, and HipA-Induced Persistence Depends on (p)ppGpp (A) Strains MG1655DhipAB 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 mg/ml), chloramphenicol (50 mg/ml), and arabinose (0.2%) without (left) or with (right) 50 mM 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 MG1655DhipBA containing the plasmids are indicated. Overnight cultures were diluted 1,000-fold in fresh rich medium with ampicillin (50 mg/ml), chloramphenicol (50 mg/ml), and 100 mM 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, DrelA, and D(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 mg/ml of ciprofloxacin (OD600 z0.5). 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
Overproduction of Glutamyl-tRNA Synthetase Counteracts HipA To investigate the molecular mechanism of HipA-mediated inhibition of cell growth, we selected for genes that, in multiple copies, would suppress HipA toxicity. We pooled a collection of plasmids obtained from the ASKA library containing most of the 4,120 E. coli genes, each cloned into the high-copy-number vector pCA24N downstream of the isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible PT5-lac promoter (Kitagawa et al., 2005). As described in the Experimental Procedures and Supplemental Information, we found only one plasmid, pCA24N::gltX, that suppressed HipA-mediated growth inhibition (Figures 2A and 2B). The gltX gene encodes glutamyl-tRNA synthetase (Kern and Lapointe, 1979), a type IB tRNA charging enzyme (Eriani et al., 1990). Our result raised the possibility that HipA inhibits translation by targeting GltX. Genes encoding EF-Tu (tufA or tufB) or other tRNA synthetases were not found in this genetic screen, consistent with the observation that none of them, when tested individually, counteracted HipA toxicity (Figures S2A and S2B). HipA Phosphorylates GltX In Vivo at Ser239 To test the possibility that HipA phosphorylates GltX, we purified GltX from a HipA-overproducing strain (see above) before and after hipA induction and analyzed the samples with MALDITOF mass spectrometry. As seen in Figure S3A, the induction of hipA increased the molecular weight (MW) of purified GltX by 80 Da, which corresponds to the substitution of hydrogen between DrelA and D(relA spoT) strains was not significant (Student’s t test; p = 0.2; n = 3). See also Figure S2.
Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 3
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
Figure 3. Phosphorylation of GltX In Vitro by HipA at Ser239 Requires tRNAGlu (A) The phosphorylation of GltX in vitro. 6 mM GltX, 0.1 mM g[32P]ATP, and 66 mM ATP were incubated with or without 0.2 mM HipA, 1.6 mM Glu, or 1.5 mM 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 mM GltX (WT, HipA treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold, and [3H]-Glu (240 counts min 1 pmol 1) and, for experiments where GltX was phosphorylated, 0.6 mM 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 mM GltX, 0.1 mM g[32P] ATP, and 66 mM ATP were incubated with or without 0.2 mM HipA, 1.6 mM Glu, or 1.5 mM tRNAGlu for 45 min. In reactions where HipA was present, HipA was added first. See also Figure S3.
with a H2PO3- group. These results suggested that GltX was phosphorylated by HipA in vivo. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that GltX was indeed phosphorylated at Ser239 (Figure S3B). Next, we analyzed the phosphorylation of GltX by HipA in vitro by using g[32P]-ATP (Figure 3A). Components were mixed prior to the addition of ATP in order to avoid the autophosphorylation of HipA (it was still autophosphorylated after the reaction with GltX was over; Figure 3A, compare lane 3 to lane 7). Non-radio-labeled ATP was added in order to suppress the hydrolysis of g[32P]-ATP by GltX. Surprisingly, negligible phosphorylation of GltX was observed (Figure 3A, lane 4). Ser239 is a part of the highly conserved flexible loop that changes conformation upon tRNAGlu binding (but not upon the binding of either Glu or ATP) (Sekine et al., 2003). We hypothesized that the phosphorylation of GltX by HipA depended on this conformational change. To test this hypothesis, we compared the phosphorylation of GltX:ATP, GltX:ATP:Glu, GltX:ATP:tRNAGlu, and GltX:ATP: tRNAGlu:Glu complexes. In accord with our proposal, the phosphorylation of GltX took place only when tRNAGlu was present in the reaction (Figure 3A, lanes 5–7).Consistently, as seen from the crystal structures, Ser239, which is shielded by several positively charged residues in the free GltX, becomes more exposed upon tRNAGlu binding (Figure 3B). Note that only catalytic amounts of HipA were required for GltX phosphorylation, consistent with the high toxicity of the protein. HipA-Dependent Phosphorylation of GltX Inhibits Its Aminoacylation Activity We tested whether HipA-mediated phosphorylation inhibited GltX catalysis of tRNA aminoacylation in vitro. Aminoacylation reactions containing saturating concentrations of ATP and [3H]-glutamate to follow the charging of tRNAGlu were assembled with native GltX and HipA-treated GltX (phosphorylated form), respectively. The resulting [3H]-Glu-tRNAGlu was precipitated on filter discs presoaked in 5% trichloroacetic acid. The discs were thoroughly washed in order to remove free [3H]-Glu, 4 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
lated Ser. The mutant GltXS239D was not phosphorylated by HipA in vitro (Figure 3D), suggesting that Ser239 was the sole site of phosphorylation by HipA. Furthermore, this mutant was inactive in the aminoacylation of tRNAGlu (Figure 3C), supporting the idea that the negative charge of aspartate at the critical Ser239 is incompatible with efficient catalytic activity of GltX. HipA-Induced Persistence Depends on (p)ppGpp HipA overproduction inhibits cell growth and simultaneously induces a high level of persistence (Korch and Hill, 2006). The inhibition of Glu-tRNAGlu caused by HipA would result in an increased concentration of uncharged tRNAGlu loading at the A site of the ribosome and, as a result, in synthesis of the alarmone (p)ppGpp by RelA (Bokinsky et al., 2013). Such a scenario would not be expected if EF-Tu was a target of HipA. We tested whether HipA-mediated persistence depended on RelA and/or SpoT, the two (p)ppGpp synthetases of E. coli. As expected, the overproduction of HipA increased the persistence of the wild-type (WT) strain by 120-fold (Figure 2C). The deletion of relA or relA and spoT resulted in a dramatic reduction of HipAmediated persistence (increases of only 4- and 2-fold, respectively). We suggest that HipA-induced persistence is mediated by (p)ppGpp. Therefore, the result supports our model in which HipA inactivates GltX, and thus generates, ‘‘hungry’’ codons that trigger (p)ppGpp production. DISCUSSION
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.
desalted, and dried, and the bound [3H]-Glu (as a measure of aminoacylation) was assessed by scintillation counting. As seen in Figure 3C, native GltX was able to aminoacylate tRNAGlu. However, when treated with HipA, the aminoacylation efficiency of GltX was dramatically reduced, suggesting that HipA inhibited GltX aminoacyl-tRNA synthetase activity (Figure 3C). These results were also obtained qualitatively by thin-layer chromatography of the glutamate released after hydrolysis of the ester bond linking it to the tRNAGlu (Figure S3C). This result is consistent with the position of Ser239 in the loop that forms part of the active center of GltX (Figure 3B; see the Discussion). To investigate the effect of phosphorylation at Ser239 further, we constructed a GltX mutant, which had Ser239, substituted with aspartate. Aspartate in this position would mimic phosphory-
HipA of E. coli is a kinase with a serine-threonine kinase fold (Correia et al., 2006) that has been proposed to inhibit translation by the phosphorylation of the essential translation factor EF-Tu (Schumacher et al., 2009). However, our results do not support this model. Instead, we present strong evidence that HipA inhibits GltX by the phosphorylation of Ser239. Ser239 belongs to the conserved KLSKR motif, which is the characteristic sequence motif of ATP-binding sites of type I aminoacyl-tRNA synthetases (Sekine et al., 2003). This motif forms a loop that participates in the binding of the catalytic ATP, which, in the absence of tRNAGlu, binds in a catalytically inactive configuration. The binding of tRNAGlu changes the conformation of the KLSKR motif, which allows the a-phosphate of ATP to align for a nucleophilic attack by Glu. The conformational change of the KLSKR motif makes Ser239 more exposed, which is consistent with our observation that HipA can transfer phosphate to Ser239 only when GltX is in complex with tRNAGlu. The critical role of the KLSKR motif in the regulation of the formation of aminoacyl-adenylate explains how the phosphorylation of Ser239 (or a negative charge of Asp in this position) may lead to the catalytic inactivation of GltX. The pleiotropic physiological effects of HipA expression was not clearly explained by previous models. Below, we discuss a molecular model explaining cellular consequences of the inhibition of GltX by HipA. In the absence of HipA, GltX is active, translation proceeds normally, and RelA is sequestered via its binding to the ribosome (Figure 4A). When HipA is present and active, GltX is inhibited by phosphorylation with the consequence that uncharged tRNAGlu accumulates. This, in turn, increases the frequency of hungry A site codons (Figure 4B). Consequently, Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 5
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
uncharged tRNAGlu enters the ribosomal A site and triggers the activation and release of RelA (English et al., 2011). RelA activation leads to an increased (p)ppGpp level that conjures the inhibition of translation, transcription, replication, and cell-wall synthesis, thereby leading to slow growth, multidrug tolerance, and persistence (Magnusson et al., 2005; Maisonneuve et al., 2013; Srivatsan and Wang, 2008). The fact that hipA induction leads to a dramatic increase in [(p)ppGpp] was shown recently by Bokinsky et al. (2013), and the mechanism behind this observation is now explained by our model. Recently, eukaryotic Lys-tRNA synthetase was shown to switch its function upon its phosphorylation in response to environmental stimulus (Ofir-Birin et al., 2013). The nonphosphorylated form acts as a common tRNA synthetase to aminoacylate tRNALys, whereas the phosphorylated one is transported to the nucleus, where it acts as a transcription factor. This raises the intriguing possibility that the phosphorylation of GltX may also switch the function of the enzyme from aminoacylation. Whether there is a switch between functions or whether phosphorylation just inactivates GltX, the ability of cells to revive from HipAinduced dormancy suggests the existence of a phosphatase that would dephosphorylate GltX. Additional investigations are required in order to test these hypotheses. EXPERIMENTAL PROCEDURES Bacterial strains and plasmids are listed in Table S1. DNA oligonucleotides are listed in Table S2. For more details, see the Supplemental Experimental Procedures. Media and Antibiotics Luria-Bertani broth was prepared as described (Clark and Maalø, 1967). When required, the medium was supplemented with 50 mg/ml ampicillin or 50 mg/ml chloramphenicol. Expression of protein from plasmids carrying pBAD promoter was induced by 0.2% arabinose and repressed by 0.2% glucose. Multicopy Suppression of HipA Toxicity by the Overproduction of Glutamyl-tRNA Synthetase A pooling mixture of the ASKA plasmid library (Kitagawa et al., 2005) was transformed into a hipBA deletion strain harboring plasmid pEG3 (pBAD30::hipA) containing an arabinose-inducible promoter. In order to induce the expression of ASKA plasmid-encoded genes, plasmids that produced colonies in the presence of IPTG were analyzed. HipA and Doc Purification HipA was purified in complex with HipB as described in Christensen-Dalsgaard et al. (2008) with a few modifications. The entire protocol is described in the Supplemental Experimental Procedures. Doc kinase was purified as described previously (Garcia-Pino et al., 2010). EF-Tu and GltX Purification Overexpressed EF-Tu or GltX were purified before or after the induction of hipA with Ni-NTA affinity chromatography as described in detail in the Supplemental Experimental Procedures. Phosphorylation of EF-Tu In Vitro HipA (0.1 mM) phosphorylation was performed in the presence or absence of 0.13 mM EF-Tu and 0.1 mM (unless otherwise specified) of g[32P]ATP (3,000 Ci/mmol; Hartmann Analytic) in a 30 ml final volume of ternary complex buffer (50 mM Tris-HCl [pH 7.4], 40 mM NH4Cl, 10 mM MgCl2, and 1 mM dithiothreitol (DTT)) for 45 min. The reaction was stopped by the addition of 1 vol Laemmli loading buffer, resolved by SDS-PAGE, and revealed by phosphorimaging (GE Healthcare).
6 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
Phosphorylation of GltX In Vitro GltX (6 mM) was mixed in aminoacylation buffer (1 mM DTT, 10 mM KCl, 16 mM ZnSO4, and 20 mM MgCl2) with 0.2 mM HipA, 66 mM ATP (nonradioactive), 0.1 mM g[32P]ATP, 1.5 mM tRNAGlu, and 1.6 mM glutamic acid. The reaction was incubated at 37 C for 45 min and was stopped by the addition of 1 volume of Laemmli buffer, resolved by SDS-PAGE, and revealed by phosphorimaging (GE Healthcare). Translation In Vitro with Cell-Free Extract In vitro translation was performed with an S30 kit (Promega) following the manufacturer’s guidelines, the exception being that 16 mg/ml total tRNA and 1 mM ATP with or without 0.1 mM HipA were added and incubated at 37 C for 10 min before the plasmid pBESTluc was added for the times indicated in Figure 1A. Preparation of Ternary Complexes for Translation In Vitro System Translation in vitro assembled from purified components was performed, and products were analyzed exactly as described in Castro-Roa and Zenkin (2012) (see the Supplemental Information for full details). HipA (0.6 mM) or Doc (0.2 mM) were added to the ternary complex or initiation complex formation reactions as described in Figure 1B. Aminoacylation Assay For quantitatively testing aminoacylation of tRNAGlu by WT GltX, phosphorylated GltX by HipA or GltX phosphomutant, an aminoacylation reaction (100 ml) containing aminoacylation buffer 13, 2 mM ATP, 0.6 mM GltX (WT, HipA-treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold, and [3H]-Glu (240 counts min 1 pmol 1) and, for experiments where GltX was phosphorylated, 0.6 mM HipA was added, and the reaction was initiated by the addition of amino acid (Kern and Lapointe, 1981). The aminoacylation reaction was initiated upon the addition of the enzyme (for WT and GltXS239D) and incubated for 3 min at 37 C. The reaction was terminated by spotting 10 ml on Whatmann 3 MM Chr filter paper presoaked in 5% TCA. The filter was immediately immersed in 15 ml of ice-cold 5% TCA for 15 min. After three washings of 15 ml of 5% TCA with incubation for 15 min in order to remove free glutamic acid, the filters were desalted in 95% ethanol for 20 min and dried overnight, and the remaining radioactivity was measured with a scintillation counter (Francklyn et al., 2008). Measurement of Persistence Persistence was measured as described previously (Maisonneuve et al., 2011) and in more detail in the Supplemental Experimental Procedures. Mass Spectrometry Analysis MALDI TOF analysis was performed as described in the Supplemental Experimental Procedures. LC MS/MS analysis was performed on purified GltX after HipA overproduction in DhipBA strain (additional information is provided in the Supplemental Experimental Procedures). SUPPLEMENTAL INFORMATION Supplemental Information contains Supplemental Experimental Procedures, three figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2013.08.045. ACKNOWLEDGMENTS We thank members of the Gerdes and Zenkin groups for stimulating discussions. We also thank Joe Gray for help with mass spectrometry analysis, Abel Garcia-Pino and Remy Loris for purified Doc, Loranne Agius for scintillation counter and Charlotte R.Knudsen for pGEX-FX-tufA plasmid coding for GST-EF-Tu. This work was funded by a European Research Council Advanced Investigator Grant [294517, ‘‘PERSIST’’] to K.G., a European Research Council Starting Grant (202994, ‘‘MTP’’), and a UK Biotechnology and Biological Sciences Research Council Grant to N.Z.
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
Received: June 14, 2013 Revised: July 29, 2013 Accepted: August 22, 2013 Published: October 3, 2013 REFERENCES Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L., and Leibler, S. (2004). Bacterial persistence as a phenotypic switch. Science 305, 1622–1625. Bigger, J.W. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation. Lancet 294, 497–500. Black, D.S., Irwin, B., and Moyed, H.S. (1994). Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. J. Bacteriol. 176, 4081–4091. Blower, T.R., Salmond, G.P., and Luisi, B.F. (2011). Balancing at survival’s edge: the structure and adaptive benefits of prokaryotic toxin-antitoxin partners. Curr. Opin. Struct. Biol. 21, 109–118. Bokinsky, G., Baidoo, E.E., Akella, S., Burd, H., Weaver, D., Alonso-Gutierrez, J., Garcı´a-Martı´n, H., Lee, T.S., and Keasling, J.D. (2013). HipA-triggered growth arrest and b-lactam tolerance in Escherichia coli are mediated by RelA-dependent ppGpp synthesis. J. Bacteriol. 195, 3173–3182. Castro-Roa, D., and Zenkin, N. (2012). In vitro experimental system for analysis of transcription-translation coupling. Nucleic Acids Res. 40, e45.
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Ofir-Birin, Y., Fang, P., Bennett, S.P., Zhang, H.M., Wang, J., Rachmin, I., Shapiro, R., Song, J., Dagan, A., Pozo, J., et al. (2013). Structural switch of lysyl-tRNA synthetase between translation and transcription. Mol. Cell 49, 30–42. Pedersen, K., Christensen, S.K., and Gerdes, K. (2002). Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Mol. Microbiol. 45, 501–510. Perla-Kajan, J., Lin, X., Cooperman, B.S., Goldman, E., Jakubowski, H., Knudsen, C.R., and Mandecki, W. (2010). Properties of Escherichia coli EFTu mutants designed for fluorescence resonance energy transfer from tRNA molecules. Protein Eng. Des. Sel. 23, 129–136. Schumacher, M.A., Piro, K.M., Xu, W., Hansen, S., Lewis, K., and Brennan, R.G. (2009). Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB. Science 323, 396–401. Sekine, S., Nureki, O., Dubois, D.Y., Bernier, S., Cheˆnevert, R., Lapointe, J., Vassylyev, D.G., and Yokoyama, S. (2003). ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding. EMBO J. 22, 676–688. Shah, D., Zhang, Z., Khodursky, A., Kaldalu, N., Kurg, K., and Lewis, K. (2006). Persisters: a distinct physiological state of E. coli. BMC Microbiol. 6, 53. Singh, R., Barry, C.E., 3rd, and Boshoff, H.I. (2010). The three RelE homologs of Mycobacterium tuberculosis have individual, drug-specific effects on bacterial antibiotic tolerance. J. Bacteriol. 192, 1279–1291. Srivatsan, A., and Wang, J.D. (2008). Control of bacterial transcription, translation and replication by (p)ppGpp. Curr. Opin. Microbiol. 11, 100–105. Va´zquez-Laslop, N., Lee, H., and Neyfakh, A.A. (2006). Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. J. Bacteriol. 188, 3494–3497. Zaher, H.S., and Green, R. (2009). Quality control by the ribosome following peptide bond formation. Nature 457, 161–166.
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Molecular Cell
Short Article Molecular Mechanism of Bacterial Persistence by HipA Elsa Germain,1,2 Daniel Castro-Roa,1,2 Nikolay Zenkin,1,* and Kenn Gerdes1,* 1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, NE2 4AX Newcastle upon Tyne, UK 2These authors contributed equally to this work *Correspondence: [email protected] (N.Z.), [email protected] (K.G.) http://dx.doi.org/10.1016/j.molcel.2013.08.045
SUMMARY
HipA of Escherichia coli is a eukaryote-like serinethreonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser239 near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNAGlu-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser239 changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of ‘‘hungry’’ codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.
INTRODUCTION Bacteria can enter a physiological state, called persistence or multidrug tolerance, in which lethal antibiotics do not kill them (Bigger, 1944). Persistence is a phenotype expressed by almost all bacteria, including major pathogens, and is believed to contribute to the intractability of chronic and relapsing infections (Levin and Rozen, 2006; Lewis, 2010). Experiments employing E. coli as the model organism indicated that persister cells form stochastically by switching into and out of slow growth (Balaban et al., 2004), a phenotype that was argued to be advantageous in changing environments (Kussell et al., 2005). Importantly, descendants of persister cells are as sensitive as their ancestors toward the bactericidal antibiotic used, demonstrating that bacterial persistence is a noninherited epigenetic characteristic (Lewis, 2010). The isolation of high-persistence (hip) mutants in E. coli indicated that persistence can have a genetic basis (Moyed and Ber-
trand, 1983). E. coli cells carrying hipA7, the most thoroughly analyzed hip mutant, exhibited a dramatic 100- to 1,000-fold increase in persistence (Korch et al., 2003). A low level of induction of hipA induced a bacteriostatic condition that could be counteracted by HipB encoded by the gene just upstream to hipA (Korch and Hill, 2006). Moreover, HipA and HipB formed a tight complex that autoregulated the transcription of the hipBA operon (Black et al., 1994). Due to these observations, it was suggested that hipBA constitutes a type II toxin-antitoxin (TA) locus (Korch et al., 2003), a suggestion that was later confirmed experimentally (Korch and Hill, 2006; Schumacher et al., 2009). Most TA loci encode two components, a toxin whose ectopic production inhibits cell growth and an antitoxin (RNA or protein) that counteracts toxin expression or activity (Blower et al., 2011; Gerdes and Maisonneuve, 2012). In type II TA loci, which are almost ubiquitous in bacteria and archaea, the antitoxins are proteins that interact with and neutralize the activity of the toxins (Gerdes et al., 2005). Most type II TA loci encode inhibitors of translation whose ectopic expression induces a static, drugtolerant condition from which the cells can be resuscitated by the induction of the antitoxin-encoding gene, consistent with a role in persistence (Correia et al., 2006; Han et al., 2010; Maisonneuve et al., 2011; Pedersen et al., 2002; Singh et al., 2010; Va´zquez-Laslop et al., 2006). E. coli has 11 canonical type II TA loci, ten of which encode toxins that inhibit translation by mRNA cleavage (Gerdes and Maisonneuve, 2012). Many type II TA operon mRNAs were significantly increased in persister cells (Keren et al., 2004; Shah et al., 2006), and the progressive deletion of these TA loci led to a gradual reduction in persistence, convincingly arguing that there is a causal connection between type II TA loci and persistence (Maisonneuve et al., 2011). Because hipA of E. coli was the first ‘‘persister’’ gene to be discovered, it has been of considerable interest in understanding how HipA inhibits cell growth and confers persistence. The ectopic induction of hipA induced growth arrest and strongly inhibited replication, transcription, and translation (Korch and Hill, 2006). Interestingly, HipA exhibits a eukaryotic serine-threonine kinase-like fold and has kinase activity (Correia et al., 2006; Schumacher et al., 2009). On the basis of in vitro experiments, it was suggested that HipA inhibits translation by the phosphorylation of EF-Tu (Schumacher et al., 2009). However, such phosphorylation did not explain the strong inhibition of replication and transcription seen after the induction of hipA (Korch and Hill, 2006; Schumacher et al., 2009). Moreover, hipA induction Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 1
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
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 mM 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 mM when HipA was added to the system: during the initiation step (blue), before formation of the ternary complex (EFTu,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 mM EF-Tu kinase Doc (CastroRoa 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 mM) after incubation for 45 min with (0.1 mM) HipA and 0.1 mM g[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.
stimulated RelA-dependent synthesis of (p)ppGpp (Bokinsky et al., 2013), thus raising the possibility that HipA has additional cellular targets. Here, we re-examine the molecular mechanism underlying HipA-induced persistence and show that the target of HipA is glutamyl-tRNA synthetase (GltX), which is inactivated by phosphorylation by HipA. Our results explain previous enigmatic physiological effects seen after the induction of hipA. RESULTS HipA Does Not Inhibit Translation by the Phosphorylation of EF-Tu HipA inactivates itself by autophosphorylation (Correia et al., 2006), which does not occur in the presence of HipB (Evdokimov et al., 2009). Therefore, we purified HipA in complex with HipB, as described previously (Christensen-Dalsgaard et al., 2008). After purification, MALDI-TOF mass spectrometry analysis revealed that more than 65% of the HipA molecules were nonphosphorylated (Figure S1A). We tested whether HipA could affect translation in vitro. As seen in Figure 1A, HipA efficiently inhibited the synthesis of luciferase in a cell-free translation system on the basis of a crude S30 cell extract. To identify the natural target of HipA, we used an in vitro translation system assembled from purified components (Castro-Roa and Zenkin, 2012). The mRNA used in this system encoded the dipeptide Met-Phe (MF) in its initial sequence. Translation was initiated by the addition of purified ribosomes, initiation factors IF-1, IF-2, IF-3, and [35S]-fMet-tRNAfMet. Then, initiated ribosomes were allowed to elongate by one codon via the addition of preformed ternary complex EF-Tu:GTP:PhetRNAPhe and the elongation factor EF-G in the presence of GTP. The short peptide products of the reaction were analyzed by thin layer electrophoresis (Castro-Roa and Zenkin, 2012; Zaher and Green, 2009). HipA was added to the reactions at three different stages: (1) during initiation complex formation (blue in Figure 1B), (2) before ternary complex formation (purple in Figure 1B), and (3) to the preformed ternary complex (TC) 2 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
(red in Figure 1B). As a positive control, we used Doc, a kinase that inhibits translation by the phosphorylation of EF-Tu (Castro-Roa et al., 2013) (Figure 1B). Surprisingly, although Doc efficiently inhibited the formation of the MF dipeptide (Figure 1B, lane 7), HipA had no effect on the reaction in any of the setups (Figure 1B, lanes 3–5). The above result contradicts the previously proposed model in which HipA inactivates translation by the phosphorylation of EFTu (Schumacher et al., 2009). Furthermore, to test this earlier model, we analyzed the phosphorylation of EF-Tu by HipA using g[32P]ATP. Given that HipA and EF-Tu migrate similarly in SDSPAGE, we used a functional GST-tagged version of EF-Tu to improve separation (Perla-Kajan et al., 2010). As seen from Figure 1C, no phosphorylation of GST-EF-Tu by HipA was observed (Figure 1C, lane 9). In contrast, GST-EF-Tu was efficiently phosphorylated by the Doc kinase (Castro-Roa et al., 2013) (Figure 1C, lane 11). Next, we decided to follow the phosphorylation state of EF-Tu in vivo. We simultaneously overproduced HipA and EF-Tu (in a strain lacking hipBA in order to avoid the neutralization of HipA by the endogenous antitoxin HipB). Then, EF-Tu purified before or after HipA overproduction was analyzed by mass spectrometry. As seen from Figure S1B (available online), we did not detect phosphorylation of EF-Tu. Altogether, our results show that HipA does not phosphorylate EF-Tu and that EF-Tu is not the target of HipA during the inactivation of translation.
Figure 2. GltX Counteracts HipA Toxicity, and HipA-Induced Persistence Depends on (p)ppGpp (A) Strains MG1655DhipAB 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 mg/ml), chloramphenicol (50 mg/ml), and arabinose (0.2%) without (left) or with (right) 50 mM 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 MG1655DhipBA containing the plasmids are indicated. Overnight cultures were diluted 1,000-fold in fresh rich medium with ampicillin (50 mg/ml), chloramphenicol (50 mg/ml), and 100 mM 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, DrelA, and D(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 mg/ml of ciprofloxacin (OD600 z0.5). 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
Overproduction of Glutamyl-tRNA Synthetase Counteracts HipA To investigate the molecular mechanism of HipA-mediated inhibition of cell growth, we selected for genes that, in multiple copies, would suppress HipA toxicity. We pooled a collection of plasmids obtained from the ASKA library containing most of the 4,120 E. coli genes, each cloned into the high-copy-number vector pCA24N downstream of the isopropyl b-D-1-thiogalactopyranoside (IPTG)-inducible PT5-lac promoter (Kitagawa et al., 2005). As described in the Experimental Procedures and Supplemental Information, we found only one plasmid, pCA24N::gltX, that suppressed HipA-mediated growth inhibition (Figures 2A and 2B). The gltX gene encodes glutamyl-tRNA synthetase (Kern and Lapointe, 1979), a type IB tRNA charging enzyme (Eriani et al., 1990). Our result raised the possibility that HipA inhibits translation by targeting GltX. Genes encoding EF-Tu (tufA or tufB) or other tRNA synthetases were not found in this genetic screen, consistent with the observation that none of them, when tested individually, counteracted HipA toxicity (Figures S2A and S2B). HipA Phosphorylates GltX In Vivo at Ser239 To test the possibility that HipA phosphorylates GltX, we purified GltX from a HipA-overproducing strain (see above) before and after hipA induction and analyzed the samples with MALDITOF mass spectrometry. As seen in Figure S3A, the induction of hipA increased the molecular weight (MW) of purified GltX by 80 Da, which corresponds to the substitution of hydrogen between DrelA and D(relA spoT) strains was not significant (Student’s t test; p = 0.2; n = 3). See also Figure S2.
Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 3
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
Figure 3. Phosphorylation of GltX In Vitro by HipA at Ser239 Requires tRNAGlu (A) The phosphorylation of GltX in vitro. 6 mM GltX, 0.1 mM g[32P]ATP, and 66 mM ATP were incubated with or without 0.2 mM HipA, 1.6 mM Glu, or 1.5 mM 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 mM GltX (WT, HipA treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold, and [3H]-Glu (240 counts min 1 pmol 1) and, for experiments where GltX was phosphorylated, 0.6 mM 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 mM GltX, 0.1 mM g[32P] ATP, and 66 mM ATP were incubated with or without 0.2 mM HipA, 1.6 mM Glu, or 1.5 mM tRNAGlu for 45 min. In reactions where HipA was present, HipA was added first. See also Figure S3.
with a H2PO3- group. These results suggested that GltX was phosphorylated by HipA in vivo. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis revealed that GltX was indeed phosphorylated at Ser239 (Figure S3B). Next, we analyzed the phosphorylation of GltX by HipA in vitro by using g[32P]-ATP (Figure 3A). Components were mixed prior to the addition of ATP in order to avoid the autophosphorylation of HipA (it was still autophosphorylated after the reaction with GltX was over; Figure 3A, compare lane 3 to lane 7). Non-radio-labeled ATP was added in order to suppress the hydrolysis of g[32P]-ATP by GltX. Surprisingly, negligible phosphorylation of GltX was observed (Figure 3A, lane 4). Ser239 is a part of the highly conserved flexible loop that changes conformation upon tRNAGlu binding (but not upon the binding of either Glu or ATP) (Sekine et al., 2003). We hypothesized that the phosphorylation of GltX by HipA depended on this conformational change. To test this hypothesis, we compared the phosphorylation of GltX:ATP, GltX:ATP:Glu, GltX:ATP:tRNAGlu, and GltX:ATP: tRNAGlu:Glu complexes. In accord with our proposal, the phosphorylation of GltX took place only when tRNAGlu was present in the reaction (Figure 3A, lanes 5–7).Consistently, as seen from the crystal structures, Ser239, which is shielded by several positively charged residues in the free GltX, becomes more exposed upon tRNAGlu binding (Figure 3B). Note that only catalytic amounts of HipA were required for GltX phosphorylation, consistent with the high toxicity of the protein. HipA-Dependent Phosphorylation of GltX Inhibits Its Aminoacylation Activity We tested whether HipA-mediated phosphorylation inhibited GltX catalysis of tRNA aminoacylation in vitro. Aminoacylation reactions containing saturating concentrations of ATP and [3H]-glutamate to follow the charging of tRNAGlu were assembled with native GltX and HipA-treated GltX (phosphorylated form), respectively. The resulting [3H]-Glu-tRNAGlu was precipitated on filter discs presoaked in 5% trichloroacetic acid. The discs were thoroughly washed in order to remove free [3H]-Glu, 4 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
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Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
lated Ser. The mutant GltXS239D was not phosphorylated by HipA in vitro (Figure 3D), suggesting that Ser239 was the sole site of phosphorylation by HipA. Furthermore, this mutant was inactive in the aminoacylation of tRNAGlu (Figure 3C), supporting the idea that the negative charge of aspartate at the critical Ser239 is incompatible with efficient catalytic activity of GltX. HipA-Induced Persistence Depends on (p)ppGpp HipA overproduction inhibits cell growth and simultaneously induces a high level of persistence (Korch and Hill, 2006). The inhibition of Glu-tRNAGlu caused by HipA would result in an increased concentration of uncharged tRNAGlu loading at the A site of the ribosome and, as a result, in synthesis of the alarmone (p)ppGpp by RelA (Bokinsky et al., 2013). Such a scenario would not be expected if EF-Tu was a target of HipA. We tested whether HipA-mediated persistence depended on RelA and/or SpoT, the two (p)ppGpp synthetases of E. coli. As expected, the overproduction of HipA increased the persistence of the wild-type (WT) strain by 120-fold (Figure 2C). The deletion of relA or relA and spoT resulted in a dramatic reduction of HipAmediated persistence (increases of only 4- and 2-fold, respectively). We suggest that HipA-induced persistence is mediated by (p)ppGpp. Therefore, the result supports our model in which HipA inactivates GltX, and thus generates, ‘‘hungry’’ codons that trigger (p)ppGpp production. DISCUSSION
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.
desalted, and dried, and the bound [3H]-Glu (as a measure of aminoacylation) was assessed by scintillation counting. As seen in Figure 3C, native GltX was able to aminoacylate tRNAGlu. However, when treated with HipA, the aminoacylation efficiency of GltX was dramatically reduced, suggesting that HipA inhibited GltX aminoacyl-tRNA synthetase activity (Figure 3C). These results were also obtained qualitatively by thin-layer chromatography of the glutamate released after hydrolysis of the ester bond linking it to the tRNAGlu (Figure S3C). This result is consistent with the position of Ser239 in the loop that forms part of the active center of GltX (Figure 3B; see the Discussion). To investigate the effect of phosphorylation at Ser239 further, we constructed a GltX mutant, which had Ser239, substituted with aspartate. Aspartate in this position would mimic phosphory-
HipA of E. coli is a kinase with a serine-threonine kinase fold (Correia et al., 2006) that has been proposed to inhibit translation by the phosphorylation of the essential translation factor EF-Tu (Schumacher et al., 2009). However, our results do not support this model. Instead, we present strong evidence that HipA inhibits GltX by the phosphorylation of Ser239. Ser239 belongs to the conserved KLSKR motif, which is the characteristic sequence motif of ATP-binding sites of type I aminoacyl-tRNA synthetases (Sekine et al., 2003). This motif forms a loop that participates in the binding of the catalytic ATP, which, in the absence of tRNAGlu, binds in a catalytically inactive configuration. The binding of tRNAGlu changes the conformation of the KLSKR motif, which allows the a-phosphate of ATP to align for a nucleophilic attack by Glu. The conformational change of the KLSKR motif makes Ser239 more exposed, which is consistent with our observation that HipA can transfer phosphate to Ser239 only when GltX is in complex with tRNAGlu. The critical role of the KLSKR motif in the regulation of the formation of aminoacyl-adenylate explains how the phosphorylation of Ser239 (or a negative charge of Asp in this position) may lead to the catalytic inactivation of GltX. The pleiotropic physiological effects of HipA expression was not clearly explained by previous models. Below, we discuss a molecular model explaining cellular consequences of the inhibition of GltX by HipA. In the absence of HipA, GltX is active, translation proceeds normally, and RelA is sequestered via its binding to the ribosome (Figure 4A). When HipA is present and active, GltX is inhibited by phosphorylation with the consequence that uncharged tRNAGlu accumulates. This, in turn, increases the frequency of hungry A site codons (Figure 4B). Consequently, Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc. 5
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
uncharged tRNAGlu enters the ribosomal A site and triggers the activation and release of RelA (English et al., 2011). RelA activation leads to an increased (p)ppGpp level that conjures the inhibition of translation, transcription, replication, and cell-wall synthesis, thereby leading to slow growth, multidrug tolerance, and persistence (Magnusson et al., 2005; Maisonneuve et al., 2013; Srivatsan and Wang, 2008). The fact that hipA induction leads to a dramatic increase in [(p)ppGpp] was shown recently by Bokinsky et al. (2013), and the mechanism behind this observation is now explained by our model. Recently, eukaryotic Lys-tRNA synthetase was shown to switch its function upon its phosphorylation in response to environmental stimulus (Ofir-Birin et al., 2013). The nonphosphorylated form acts as a common tRNA synthetase to aminoacylate tRNALys, whereas the phosphorylated one is transported to the nucleus, where it acts as a transcription factor. This raises the intriguing possibility that the phosphorylation of GltX may also switch the function of the enzyme from aminoacylation. Whether there is a switch between functions or whether phosphorylation just inactivates GltX, the ability of cells to revive from HipAinduced dormancy suggests the existence of a phosphatase that would dephosphorylate GltX. Additional investigations are required in order to test these hypotheses. EXPERIMENTAL PROCEDURES Bacterial strains and plasmids are listed in Table S1. DNA oligonucleotides are listed in Table S2. For more details, see the Supplemental Experimental Procedures. Media and Antibiotics Luria-Bertani broth was prepared as described (Clark and Maalø, 1967). When required, the medium was supplemented with 50 mg/ml ampicillin or 50 mg/ml chloramphenicol. Expression of protein from plasmids carrying pBAD promoter was induced by 0.2% arabinose and repressed by 0.2% glucose. Multicopy Suppression of HipA Toxicity by the Overproduction of Glutamyl-tRNA Synthetase A pooling mixture of the ASKA plasmid library (Kitagawa et al., 2005) was transformed into a hipBA deletion strain harboring plasmid pEG3 (pBAD30::hipA) containing an arabinose-inducible promoter. In order to induce the expression of ASKA plasmid-encoded genes, plasmids that produced colonies in the presence of IPTG were analyzed. HipA and Doc Purification HipA was purified in complex with HipB as described in Christensen-Dalsgaard et al. (2008) with a few modifications. The entire protocol is described in the Supplemental Experimental Procedures. Doc kinase was purified as described previously (Garcia-Pino et al., 2010). EF-Tu and GltX Purification Overexpressed EF-Tu or GltX were purified before or after the induction of hipA with Ni-NTA affinity chromatography as described in detail in the Supplemental Experimental Procedures. Phosphorylation of EF-Tu In Vitro HipA (0.1 mM) phosphorylation was performed in the presence or absence of 0.13 mM EF-Tu and 0.1 mM (unless otherwise specified) of g[32P]ATP (3,000 Ci/mmol; Hartmann Analytic) in a 30 ml final volume of ternary complex buffer (50 mM Tris-HCl [pH 7.4], 40 mM NH4Cl, 10 mM MgCl2, and 1 mM dithiothreitol (DTT)) for 45 min. The reaction was stopped by the addition of 1 vol Laemmli loading buffer, resolved by SDS-PAGE, and revealed by phosphorimaging (GE Healthcare).
6 Molecular Cell 52, 1–7, October 24, 2013 ª2013 Elsevier Inc.
Phosphorylation of GltX In Vitro GltX (6 mM) was mixed in aminoacylation buffer (1 mM DTT, 10 mM KCl, 16 mM ZnSO4, and 20 mM MgCl2) with 0.2 mM HipA, 66 mM ATP (nonradioactive), 0.1 mM g[32P]ATP, 1.5 mM tRNAGlu, and 1.6 mM glutamic acid. The reaction was incubated at 37 C for 45 min and was stopped by the addition of 1 volume of Laemmli buffer, resolved by SDS-PAGE, and revealed by phosphorimaging (GE Healthcare). Translation In Vitro with Cell-Free Extract In vitro translation was performed with an S30 kit (Promega) following the manufacturer’s guidelines, the exception being that 16 mg/ml total tRNA and 1 mM ATP with or without 0.1 mM HipA were added and incubated at 37 C for 10 min before the plasmid pBESTluc was added for the times indicated in Figure 1A. Preparation of Ternary Complexes for Translation In Vitro System Translation in vitro assembled from purified components was performed, and products were analyzed exactly as described in Castro-Roa and Zenkin (2012) (see the Supplemental Information for full details). HipA (0.6 mM) or Doc (0.2 mM) were added to the ternary complex or initiation complex formation reactions as described in Figure 1B. Aminoacylation Assay For quantitatively testing aminoacylation of tRNAGlu by WT GltX, phosphorylated GltX by HipA or GltX phosphomutant, an aminoacylation reaction (100 ml) containing aminoacylation buffer 13, 2 mM ATP, 0.6 mM GltX (WT, HipA-treated or GltXS239D), 0.2 mg/ml tRNA, 100 mM glutamate cold, and [3H]-Glu (240 counts min 1 pmol 1) and, for experiments where GltX was phosphorylated, 0.6 mM HipA was added, and the reaction was initiated by the addition of amino acid (Kern and Lapointe, 1981). The aminoacylation reaction was initiated upon the addition of the enzyme (for WT and GltXS239D) and incubated for 3 min at 37 C. The reaction was terminated by spotting 10 ml on Whatmann 3 MM Chr filter paper presoaked in 5% TCA. The filter was immediately immersed in 15 ml of ice-cold 5% TCA for 15 min. After three washings of 15 ml of 5% TCA with incubation for 15 min in order to remove free glutamic acid, the filters were desalted in 95% ethanol for 20 min and dried overnight, and the remaining radioactivity was measured with a scintillation counter (Francklyn et al., 2008). Measurement of Persistence Persistence was measured as described previously (Maisonneuve et al., 2011) and in more detail in the Supplemental Experimental Procedures. Mass Spectrometry Analysis MALDI TOF analysis was performed as described in the Supplemental Experimental Procedures. LC MS/MS analysis was performed on purified GltX after HipA overproduction in DhipBA strain (additional information is provided in the Supplemental Experimental Procedures). SUPPLEMENTAL INFORMATION Supplemental Information contains Supplemental Experimental Procedures, three figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2013.08.045. ACKNOWLEDGMENTS We thank members of the Gerdes and Zenkin groups for stimulating discussions. We also thank Joe Gray for help with mass spectrometry analysis, Abel Garcia-Pino and Remy Loris for purified Doc, Loranne Agius for scintillation counter and Charlotte R.Knudsen for pGEX-FX-tufA plasmid coding for GST-EF-Tu. This work was funded by a European Research Council Advanced Investigator Grant [294517, ‘‘PERSIST’’] to K.G., a European Research Council Starting Grant (202994, ‘‘MTP’’), and a UK Biotechnology and Biological Sciences Research Council Grant to N.Z.
Please cite this article in press as: Germain et al., Molecular Mechanism of Bacterial Persistence by HipA, Molecular Cell (2013), http://dx.doi.org/ 10.1016/j.molcel.2013.08.045
Molecular Cell HipA Inhibits Glutamyl-tRNA Synthetase
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